Recceing Oligomers, Polymers and Other Cryoprotective Agents

**Chapter 1**

**Abstract**

improved with extracellular CPA.

**1. Introduction**

**3**

physical approach, differential scanning calorimetry

The Use of Chitooligosaccharides

Concept and First Answers from

DSC Thermal Analysis

in Cryopreservation: Discussion of

*Hugo Desnos, Pierre Bruyère, Magda Teixeira, Loris Commin,*

The use of dimethyl sulfoxide (Me2SO) as a cryoprotectant agent (CPA) is controversial. Indeed, this cryoprotectant agent (CPA) is cytotoxic and potentially mutagenic. Therefore, other cryoprotectants must be used to reduce the proportion of Me2SO in slow-freezing solutions. In this chapter, we propose to present the first evaluation of new non-penetrating cryoprotectants: the chitooligosaccharides (COS). These molecules are chitosan oligomers, which are biocompatible, antioxidant, and bacteriostatic. We first review the use of saccharides through cryopreservation processes. We question the possibility to reduce penetrating CPA during slow-freezing procedures. We propose to use COS as extracellular CPA to reduce the use of Me2SO. We question the biocompatibility of COS on mouse embryos through the analysis of the cells' development. Next, we evaluate these molecules in slow-freezing solutions with a reduced quantity of Me2SO. Our experimental approach is a physical method often used to characterize slow-freezing solutions. Differential scanning calorimetry (DSC) allows to evaluate the crystallization and melting processes, the amount of crystallized water, and the equilibrium temperature and consequently to evaluate the impact of different cryoprotectants. This study gives a better understanding on how slow-freezing protocols could be

**Keywords:** slow-freezing improvement, chitooligosaccharides, dimethyl sulfoxide,

Despite the progress achieved these last decades, the improvement of cryopreservation procedures is still desired by the scientific community [1, 2]. A possible improvement is the decrease of the penetrating cryoprotective agents (CPA). The small and osmotically active penetrating molecules used as penetrating CPA in cryopreservation present risks of cytotoxicity for cells [3–6]. These molecules penetrate easily into tissues and, after impregnation, are difficult to fully extract from

*Gérard Louis, Stephane Trombotto, Amani Moussa,*

*Laurent David, Samuel Buff and Anne Baudot*

## **Chapter 1**

## The Use of Chitooligosaccharides in Cryopreservation: Discussion of Concept and First Answers from DSC Thermal Analysis

*Hugo Desnos, Pierre Bruyère, Magda Teixeira, Loris Commin, Gérard Louis, Stephane Trombotto, Amani Moussa, Laurent David, Samuel Buff and Anne Baudot*

## **Abstract**

The use of dimethyl sulfoxide (Me2SO) as a cryoprotectant agent (CPA) is controversial. Indeed, this cryoprotectant agent (CPA) is cytotoxic and potentially mutagenic. Therefore, other cryoprotectants must be used to reduce the proportion of Me2SO in slow-freezing solutions. In this chapter, we propose to present the first evaluation of new non-penetrating cryoprotectants: the chitooligosaccharides (COS). These molecules are chitosan oligomers, which are biocompatible, antioxidant, and bacteriostatic. We first review the use of saccharides through cryopreservation processes. We question the possibility to reduce penetrating CPA during slow-freezing procedures. We propose to use COS as extracellular CPA to reduce the use of Me2SO. We question the biocompatibility of COS on mouse embryos through the analysis of the cells' development. Next, we evaluate these molecules in slow-freezing solutions with a reduced quantity of Me2SO. Our experimental approach is a physical method often used to characterize slow-freezing solutions. Differential scanning calorimetry (DSC) allows to evaluate the crystallization and melting processes, the amount of crystallized water, and the equilibrium temperature and consequently to evaluate the impact of different cryoprotectants. This study gives a better understanding on how slow-freezing protocols could be improved with extracellular CPA.

**Keywords:** slow-freezing improvement, chitooligosaccharides, dimethyl sulfoxide, physical approach, differential scanning calorimetry

## **1. Introduction**

Despite the progress achieved these last decades, the improvement of cryopreservation procedures is still desired by the scientific community [1, 2]. A possible improvement is the decrease of the penetrating cryoprotective agents (CPA). The small and osmotically active penetrating molecules used as penetrating CPA in cryopreservation present risks of cytotoxicity for cells [3–6]. These molecules penetrate easily into tissues and, after impregnation, are difficult to fully extract from

biological systems. Among the different penetrating CPA, Me2SO is a penetrating CPA especially efficient to promote successful cryopreservation. However, the Me2SO molecule presents risks of toxicity to biological materials [7–14]. It is also suspected to be an intercalator of DNA [15]. In this chapter, we propose to study the possibility to reduce the use of the Me2SO molecule while maintaining the survival rate of cells in slow-freezing cryopreservation.

explained (Part 4). Then, COS synthesis is presented from a physicochemical point of view (Part 5). The biocompatibility of COS on mouse embryos is questioned (Part 6), and a first understanding of the action of COS as cryoprotectants is quantified by DSC analysis (Part 7), before a conclusion on the possibility of using

*The Use of Chitooligosaccharides in Cryopreservation: Discussion of Concept and First…*

The cryoprotective role of saccharide compounds (mono-, di-, tri-, oligo-, or polysaccharides) is known for a long time. Some of them are secreted by certain cold-blooded organisms that withstand harsh winters [22, 23]. Several saccharides are water soluble, have a high osmotic effect, and have many influences on the solutions' properties: they can bind part of the water and make it non-crystallizable [24–29]; their action on viscosity [30, 31] has been highlighted, as well as their ability to disrupt the organization of water molecules in the liquid phase [32–35]. In a mixture of "H2O/saccharide compound," the proportion of water has an influence on the properties of the saccharide compound and therefore on the properties of the

These compounds are known to be unable to penetrate passively through most cell membranes. From the cryoprotective point of view, they act outside the cells, giving them limited action on cell protection. This is the reason why the standard in cryopreservation procedures remains the massive use of penetrating CPA that possess a high osmotic effect in both extra- and intracellular solutions. However, the beneficial use of saccharides to substitute penetrating CPA compounds has been proven in recent years. Indeed, saccharides have been used for cell cryopreservation in slow-freezing [37–51] or vitrification procedures [16–18, 39, 52–55]. A nonexhaustive overview from the literature of the use of the saccharides is displayed in

Using empirical methods, it has been shown that these compounds allow the improvement of the post-cryopreservation survival rates of certain cell types [3, 37,

• Meryman was the first to propose that the presence of large polymers in solutions prevents the risks of osmotic shocks (when the penetrating CPA are released from the intracellular medium) as well as the denaturation of cell

• In cryopreservation of spermatozoa, small saccharides are often used as

**Monosaccharides Disaccharides Trisaccharide** Glucose [56–63] Sucrose [61, 64–67] Raffinose [14] Fructose [14] Maltose [35] **Polysaccharides**

substituents for penetrating CPA [42, 49, 50, 61]. The advanced argument of their use is that they increase the viscosity, increase the glass-forming tendency, and protect the cellular lipid membranes [42, 61, 82].

Sorbitol [14] Trehalose [16, 35, 39–43, 49–51, 61, 68–79] Dextran [1, 2, 17, 18, 54, 67, 80, 81]

Galactose [64] Ficoll [1, 17, 18, 54, 64, 66, 81]

*Non-exhaustive directory from literature of saccharides used in cryopreservation procedures.*

38, 44–48, 82]. Some authors have discussed the benefits of using them:

**2. State of the art: the use of saccharides in cryopreservation**

these compounds as non-penetrating CPA.

*DOI: http://dx.doi.org/10.5772/intechopen.89162*

mixture [29, 36].

**Table 1**.

membranes [83].

Mannitol [14]

**Table 1.**

**5**

A challenge of modern cell cryopreservation is to propose procedures in which CPA concentration is as little as possible:


We assume, however, that a good combination of penetrating and nonpenetrating CPA and an adaptation of the slow-freezing protocols could provide satisfactory survival rates, despite a reduction in the initial proportion of Me2SO used. This chapter investigates an alternative to the current slow-freezing procedures using Me2SO and proposes procedures where the initial and necessary amount of Me2SO is lowered.

Decreasing the Me2SO proportion in solution will modify the couple "protocol/ solution" within the cryopreservation procedure since it is necessary to counterbalance the loss of the cryoprotective effect of the removed Me2SO molecules. To that end, this chapter studies the possibility to use chitooligosaccharide (COS) compounds in solution. The role of extracellular CPA is discussed, and the effect of the COS in solution is thermodynamically evaluated.

Even if common characteristics of various CPA can be defined, the substances currently used as CPA have different chemical structures and sizes, so it is still difficult to predict the cryoprotective properties of substances from their basic chemical structures. The modes of action of cryoprotectants have not yet been fully elucidated. Thus, the development of effective cryopreservation solutions remains primarily based on empirical considerations. Consequently, studying the potential cryoprotective characteristics of new compounds serves a dual purpose: (1) propose new cryoprotectants and (2) understand the mechanisms of cryoprotection. It makes possible to better classify the molecules according to their modes of action regarding cellular cryoprotection.

This chapter aims to assess the use of COS to fulfill part of the cryoprotective role of the Me2SO. It presents first, from the cellular cryopreservation point of view, the interesting properties of saccharides (Part 2). It discusses next the optimization of the procedures required to replace a part of the penetrating CPA by using nonpenetrating saccharides (Part 3). The use of COS to realize this optimization is

*The Use of Chitooligosaccharides in Cryopreservation: Discussion of Concept and First… DOI: http://dx.doi.org/10.5772/intechopen.89162*

explained (Part 4). Then, COS synthesis is presented from a physicochemical point of view (Part 5). The biocompatibility of COS on mouse embryos is questioned (Part 6), and a first understanding of the action of COS as cryoprotectants is quantified by DSC analysis (Part 7), before a conclusion on the possibility of using these compounds as non-penetrating CPA.

## **2. State of the art: the use of saccharides in cryopreservation**

The cryoprotective role of saccharide compounds (mono-, di-, tri-, oligo-, or polysaccharides) is known for a long time. Some of them are secreted by certain cold-blooded organisms that withstand harsh winters [22, 23]. Several saccharides are water soluble, have a high osmotic effect, and have many influences on the solutions' properties: they can bind part of the water and make it non-crystallizable [24–29]; their action on viscosity [30, 31] has been highlighted, as well as their ability to disrupt the organization of water molecules in the liquid phase [32–35]. In a mixture of "H2O/saccharide compound," the proportion of water has an influence on the properties of the saccharide compound and therefore on the properties of the mixture [29, 36].

These compounds are known to be unable to penetrate passively through most cell membranes. From the cryoprotective point of view, they act outside the cells, giving them limited action on cell protection. This is the reason why the standard in cryopreservation procedures remains the massive use of penetrating CPA that possess a high osmotic effect in both extra- and intracellular solutions. However, the beneficial use of saccharides to substitute penetrating CPA compounds has been proven in recent years. Indeed, saccharides have been used for cell cryopreservation in slow-freezing [37–51] or vitrification procedures [16–18, 39, 52–55]. A nonexhaustive overview from the literature of the use of the saccharides is displayed in **Table 1**.

Using empirical methods, it has been shown that these compounds allow the improvement of the post-cryopreservation survival rates of certain cell types [3, 37, 38, 44–48, 82]. Some authors have discussed the benefits of using them:



### **Table 1.**

*Non-exhaustive directory from literature of saccharides used in cryopreservation procedures.*

biological systems. Among the different penetrating CPA, Me2SO is a penetrating CPA especially efficient to promote successful cryopreservation. However, the Me2SO molecule presents risks of toxicity to biological materials [7–14]. It is also suspected to be an intercalator of DNA [15]. In this chapter, we propose to study the possibility to reduce the use of the Me2SO molecule while maintaining the survival

A challenge of modern cell cryopreservation is to propose procedures in which

• In vitrification procedures, the techniques seek to obtain the glassy state with very small amounts of penetrating CPA (less than 25% (w/w)) by replacing them with non-penetrating CPA [3, 16–18]. Accordingly, the cooling must be applied as quickly as possible so that the cells do not suffer the damage associated with excessive dehydration and/or volume loss at room

temperature. They also require the use of particularly small sample volumes (<1 μL) to reach high cooling rate that will prevent ice crystallization. These procedures are particularly delicate and require a certain dexterity of the

• In slow-freezing procedures, reducing the initial amount of penetrating CPA significantly increases the stresses and risks experienced by cells during cryopreservation. It is therefore difficult to propose an excessive reduction in the initial concentration of penetrating CPA. Indeed, it has been theoretically demonstrated, through mathematical modeling, that it is impossible to cryopreserve cells with initial Me2SO concentrations below 1 mol.L<sup>1</sup> [21].

We assume, however, that a good combination of penetrating and nonpenetrating CPA and an adaptation of the slow-freezing protocols could provide satisfactory survival rates, despite a reduction in the initial proportion of Me2SO used. This chapter investigates an alternative to the current slow-freezing procedures using Me2SO and proposes procedures where the initial and necessary amount

Decreasing the Me2SO proportion in solution will modify the couple "protocol/ solution" within the cryopreservation procedure since it is necessary to counterbalance the loss of the cryoprotective effect of the removed Me2SO molecules. To that end, this chapter studies the possibility to use chitooligosaccharide (COS) compounds in solution. The role of extracellular CPA is discussed, and the effect of the

Even if common characteristics of various CPA can be defined, the substances currently used as CPA have different chemical structures and sizes, so it is still difficult to predict the cryoprotective properties of substances from their basic chemical structures. The modes of action of cryoprotectants have not yet been fully elucidated. Thus, the development of effective cryopreservation solutions remains primarily based on empirical considerations. Consequently, studying the potential cryoprotective characteristics of new compounds serves a dual purpose: (1) propose new cryoprotectants and (2) understand the mechanisms of cryoprotection. It makes possible to better classify the molecules according to their modes of action

This chapter aims to assess the use of COS to fulfill part of the cryoprotective role of the Me2SO. It presents first, from the cellular cryopreservation point of view, the interesting properties of saccharides (Part 2). It discusses next the optimization of the procedures required to replace a part of the penetrating CPA by using nonpenetrating saccharides (Part 3). The use of COS to realize this optimization is

rate of cells in slow-freezing cryopreservation.

*Cryopreservation - Current Advances and Evaluations*

CPA concentration is as little as possible:

experimenter [19, 20].

of Me2SO is lowered.

COS in solution is thermodynamically evaluated.

regarding cellular cryoprotection.

**4**

• In cryopreservation by vitrification, Kuleshova et al. [17] proposed to replace a large portion of penetrating CPA by large extracellular polysaccharides (Ficoll and dextran).

improves sperm motility after cryopreservation [88]. Finally, it has been proposed to create hydrogel-based microspheres to encapsulate cellular

membranes. By interacting with cell membranes, saccharides have an effect on the fluidity of lipidic membranes [82, 83]. Interactions between membrane phospholipids and saccharides in solution have also been advanced as a method

Saccharidic CPA are used in different ways depending on their physicochemical characteristics. In general, the polysaccharides are added in solution to increase the viscosity while having a weak osmotic activity. On the contrary, the small saccharide molecules (such as glucose) are added in solution to have high osmotic activity within the solution (this favors and induces the cellular dehydration), without

**3. Strategy for improving slow-freezing cryopreservation procedures**

[21]. But these models studied this reduction without trying to use other CPA compounds with completely different properties than those currently used. Me2SO is certainly one of the best compounds to fulfill the roles that are required to a CPA (apart from its cytotoxicity), but it is conceivable that a combination of different

eral assumptions were considered, based on results published in the literature:

a. The slow-freezing procedures have been optimized according to some

Other combinations of the parameters may be better than the currently used. The search for new CPA is still ongoing, and the cryopreservation procedures are better understood. The discovery of intracellular vitreous transitions [87], the control of the IRI process [92], the development of new cooling or warming

techniques [93], etc. may suggest that optimization of the procedure is still possible.

cryopreservation solution is considered in order to find a formulation offering

It has been shown that the proportion of penetrating CPA necessary to lead to a

cellular vitrification can be reduced if there is a high cellular dehydration [16]. Indeed, the intracellular cytoplasmic medium has a great glass-forming tendency because of the presence of many macromolecules [87, 94–96]. The presence in intracellular solution of a large proportion of proteins and other organic compounds (macromolecules), accompanied by dehydration of the medium, is sufficient to promote vitrification [87, 94]. In addition, experiments have shown the existence of

colloidal glass transitions in cells during their dehydration [96, 97]. These

transitions can promote intracellular vitrification and potentially protect cells (since

b. The optimization of the proportion of penetrating CPA in the

good vitrification conditions for the remaining solution.

Models showed that the improvement of cryopreservation procedures is impossible if it implies an excessive reduction in the use of penetrating CPA (<1 mol.L<sup>1</sup>

To reduce the use of Me2SO in slow-freezing cryopreservation procedures, sev-

)

• Saccharides promote the lipid transition of phospholipids from cell

*The Use of Chitooligosaccharides in Cryopreservation: Discussion of Concept and First…*

of promoting the success of cryopreservation procedures [90, 91].

systems in small volumes [89].

*DOI: http://dx.doi.org/10.5772/intechopen.89162*

changing the viscosity too much.

**3.1 Foundations of the strategy**

**7**

CPA may also be compatible with cell survival.

parameters, but new optimizations are possible.


From these readings, mechanisms can be advanced to explain how the addition of saccharides in solution improves cell survival:


*The Use of Chitooligosaccharides in Cryopreservation: Discussion of Concept and First… DOI: http://dx.doi.org/10.5772/intechopen.89162*

improves sperm motility after cryopreservation [88]. Finally, it has been proposed to create hydrogel-based microspheres to encapsulate cellular systems in small volumes [89].

• Saccharides promote the lipid transition of phospholipids from cell membranes. By interacting with cell membranes, saccharides have an effect on the fluidity of lipidic membranes [82, 83]. Interactions between membrane phospholipids and saccharides in solution have also been advanced as a method of promoting the success of cryopreservation procedures [90, 91].

Saccharidic CPA are used in different ways depending on their physicochemical characteristics. In general, the polysaccharides are added in solution to increase the viscosity while having a weak osmotic activity. On the contrary, the small saccharide molecules (such as glucose) are added in solution to have high osmotic activity within the solution (this favors and induces the cellular dehydration), without changing the viscosity too much.

## **3. Strategy for improving slow-freezing cryopreservation procedures**

## **3.1 Foundations of the strategy**

• In cryopreservation by vitrification, Kuleshova et al. [17] proposed to replace a large portion of penetrating CPA by large extracellular polysaccharides (Ficoll

• In slow-freezing cryopreservation, saccharides have also been used [84]. For the cryopreservation of human or mouse embryos, Dumoulin et al. [81] highlighted the ability of large molecules (e.g., dextran) to reduce damage to zona pellucida when added in small amounts (i.e., less than 10% (w/v)). For the cryopreservation of cat embryos, Gómez et al. [67] were able to obtain interesting survival rates (>80%) by combining propylene glycol (1.4 M), sucrose (0.125 M), and dextran 10% (w/w). Slow-freezing procedures for some weak cellular systems have been proposed, without recourse to CPA penetrants [43]. This strategy is based on the induction, prior to ice formation, of cellular dehydration using osmotically active extracellular compounds (e.g.,

• For the slow-freezing cryopreservation of human red blood cells, Bailey et al. [85] reported the possibility to reduce the use of Me2SO from 10% (w/w) to

recrystallization inhibition (IRI) activity could be seen, the interaction of those copolymers with cells' membranes should be the reason that increases the cell

From these readings, mechanisms can be advanced to explain how the addition

• Saccharides have a cryoscopic influence in the extracellular medium [86]. It reduces the damage associated with the formation and growth of the crystalline

• Saccharides promote cell dehydration on cooling [17]. This aspect is ambivalent because, on the one hand, it allows to promote intracellular vitrification, but on the other hand, it increases the risks associated with

• Saccharides reduce the cellular rehydration during warming [19, 83]. By

having an osmotic effect from the extracellular medium, they limit the osmotic gap (appeared during cooling, due to the difference between the intra- and the extracellular vitrification temperature [87]) between intra- and extracellular media. During warming and during the ice melting, saccharides guarantee a

• Saccharides participate in the evolution of extracellular viscosity. This reduces the kinetics of crystal growth and favors the deviation of the system from its equilibrium position. This effect has the consequence of improving the

• Saccharides allow to "encapsulate" cells. Thus, the presence of large molecules in solution protects the zona pellucida of embryos during slow-freezing procedures [81]. Likewise, the presence of these large molecules can move the cells away from the ice in the overconcentrated amorphous phase [82]. It has been also reported that the presence of mono- or disaccharides in solution

2.5% (w/w) through the use of high concentration of copolymers (polyampholytes). These authors concluded that because no ice

and dextran).

*Cryopreservation - Current Advances and Evaluations*

trehalose).

recovery.

phase.

**6**

cellular dehydration.

of saccharides in solution improves cell survival:

smoother return of water into the cells [19, 83].

extracellular vitreous state achievement [17].

Models showed that the improvement of cryopreservation procedures is impossible if it implies an excessive reduction in the use of penetrating CPA (<1 mol.L<sup>1</sup> ) [21]. But these models studied this reduction without trying to use other CPA compounds with completely different properties than those currently used. Me2SO is certainly one of the best compounds to fulfill the roles that are required to a CPA (apart from its cytotoxicity), but it is conceivable that a combination of different CPA may also be compatible with cell survival.

To reduce the use of Me2SO in slow-freezing cryopreservation procedures, several assumptions were considered, based on results published in the literature:

a. The slow-freezing procedures have been optimized according to some parameters, but new optimizations are possible.

Other combinations of the parameters may be better than the currently used. The search for new CPA is still ongoing, and the cryopreservation procedures are better understood. The discovery of intracellular vitreous transitions [87], the control of the IRI process [92], the development of new cooling or warming techniques [93], etc. may suggest that optimization of the procedure is still possible.

b. The optimization of the proportion of penetrating CPA in the cryopreservation solution is considered in order to find a formulation offering good vitrification conditions for the remaining solution.

It has been shown that the proportion of penetrating CPA necessary to lead to a cellular vitrification can be reduced if there is a high cellular dehydration [16]. Indeed, the intracellular cytoplasmic medium has a great glass-forming tendency because of the presence of many macromolecules [87, 94–96]. The presence in intracellular solution of a large proportion of proteins and other organic compounds (macromolecules), accompanied by dehydration of the medium, is sufficient to promote vitrification [87, 94]. In addition, experiments have shown the existence of colloidal glass transitions in cells during their dehydration [96, 97]. These transitions can promote intracellular vitrification and potentially protect cells (since cell cytoplasm is often heavily loaded with large molecules, it can easily vitrify [17, 59, 96]). In a vitrification procedure, using successive baths of osmotic equilibration, the cells are placed in solutions containing penetrating CPA. It was proposed by Kuleshova et al. [17] to replace, in those procedures, a large proportion of penetrating CPA with non-penetrating polymers (PVP, dextran, Ficoll, etc.). In the last osmotic equilibration bath, non-penetrating CPA (sugars or polymers) are added to promote cell dehydration just before the plunge into liquid nitrogen (LN2). The osmotic effect of these non-penetrating CPA promotes the vitrification of the intracellular medium and ensures the success of the procedure with a lower amount of penetrating CPA [16]. By adaptation to slow-freezing procedures, we assume that it is possible to force the intracellular colloidal glass transition to the correct temperature by suitably combining temperature lowering and cellular dehydration. However, cellular dehydration is not possible over long periods when the temperature remains high. During a vitrification procedure, the dehydration time is short (primarily applied during the last osmotic bath, a few seconds before the plunge into LN2). During slow-freezing, if the dehydration intervenes too extremely, it could lead to cellular collapse [83, 98, 99]. To solve this problem, we rely on the work of Mazur et al. [100] who proposed an alternative to conventional cellular cryopreservation protocols by rapidly bringing the cells to a temperature low enough to make the effects of cell dehydration less deleterious. This temperature must, however, remain above the nucleation temperature of the intracellular medium. A temperature stabilization is then carried out at this intermediate temperature to allow the equilibration of the media. Then, the cooling continues at a slow cooling rate until the plunge into LN2. This type of protocol is called "rapid cooling interrupted" [100]. It should be noted that this protocol also possesses a seeding step in order to control the extracellular ice growth. We assume that, when using this type of protocol, it would be possible to allow faster cell dehydration already biologically tolerated in the case of vitrification procedures.

the CPA penetration. This rate deals with the two major risks of the slow-freezing procedures, the "solute effect," which occurs at low cooling rate, and the intracellular

*The Use of Chitooligosaccharides in Cryopreservation: Discussion of Concept and First…*

etc. Thus, penetrating CPA play a cryoprotective role that cannot be fully

reproduced in the extracellular environment by non-penetrating CPA. Moreover, the stresses and risks associated with the ice formation must be limited or prevented until the plunge to LN2. It is therefore necessary to continue to use penetrating CPA, but their amount in the initial solution should be limited to reduce cytotoxicity. In addition to the presence of penetrating CPA, the vitrification of the extracellular remaining solution depends on the presence of non-penetrating CPA that limit the evolution of penetrating CPA concentrations and reduce the risks associated with the ice formation in solution. The reduction of penetrating CPA implies to control the damages related to the ice formation in solution by replacing a large part of the initial extracellular water by non-crystallizable substances, which offer favorable conditions for cryopreservation. However, the biocompatibility of these com-

Because the proportion of CPA in the intracellular medium has a lighter impact than the cellular dehydration on the intracellular vitrification [16, 87, 94–96], we assume that the proportion of penetrating CPA may be limited. In that case, it is then necessary to reduce the deleterious effects related to the cellular dehydration by using non-penetrating CPA that are not highly osmotically active and whose molar concentrations slightly change during cooling (i.e., compounds with a large molar mass). It is also necessary to employ the "fast interrupt protocol" by suitably combining dehydration and lowering temperature so that the colloidal intracellular

Finally, it is necessary to verify that the extracellular remaining solution can vitrify easily so that the cryopreservation procedure can be interrupted at a higher plunge temperature into LN2. Thus, the use of cryostabilizer CPA that facilitate the achievement of the vitreous state, without binding a significant amount of water, may promote the stabilization of the system "ice/remaining solution" (by reducing

We chose to study the possibility to cryopreserve mouse embryos with 5% (v/v) of Me2SO in the initial solution, supplemented with non-penetrating CPA (COS).

The idea of using COS compounds in cell cryopreservation is supported by:

• An absence of toxicity, an ability to degrade without toxic residues, and a

The addition of penetrating CPA in a solution, in a reasonable proportion (> 1 mol. L<sup>1</sup> for slow-freezing and > 3 mol.L<sup>1</sup> for vitrification), is considered essential for the survival of mammalian embryo type cells [21]. Very few studies have been published on attempts to reduce the amount of Me2SO in solution during slow-freezing cryopreservation [43, 84]. This strategy was evaluated by relying on modeling arguments, showing that it seems complicated to reduce this initial proportion of penetrating CPA [21]. We make a proposal for slow-freezing procedure optimization using a reduced initial proportion of penetrating CPA, supplemented with non-penetrating CPA. The decrease of the initial proportion of penetrating CPA implies an increase in the amount of crystallizable water, a stronger evolution of the crystal/liquid ratio in the solution with temperature, a higher amount of penetrating CPA needed to diffuse through the membrane, more risks of contact between cells and ice crystals,

nucleation, which occurs at high cooling rate [101].

*DOI: http://dx.doi.org/10.5772/intechopen.89162*

pounds on the cellular systems must be guaranteed.

**4. The proposition to use COS**

biocompatibility [102].

**9**

transition occurs before cell dehydration becomes too important.

the risk of recrystallization of the intercrystalline remaining solution).

c. The use of non-penetrating CPA can allow reducing the necessary proportion of penetrating CPA by optimizing the procedure of cooling and warming.

For a slow-freezing procedure, in which ice formation is allowed and desired, the osmotic effect of a non-penetrating CPA becomes increasingly important as the proportion of water in the remaining extracellular medium decreases. Conversely, in the intracellular medium, it is mainly the penetrating CPA that participates in the osmotic balance. There is therefore a gap between these two media in the osmolality evolution, which accentuates cellular dehydration and increases cell contraction at a given cooling rate. The use of an osmotically active non-penetrating CPA in solution may induce a significant change in the osmolality difference between the intra- and the extracellular media.

## **3.2 The elements of the proposed optimization**

The toxicity of the cryopreservation solutions is related to the CPA concentration reached in the remaining solution to enable the vitrification of the intracellular medium. Moreover, to protect the cells, the vitrification must also be carried out in the noncrystalline extracellular medium in contact with the cells. Intracellular vitrification is dependent on the presence of penetrating CPA and on the cellular dehydration. In consequence, the intracellular vitrification is dependent on the diffusion of materials across the cellular membrane. That is why cryopreservation procedures are time dependent. Indeed, an ideal cooling rate exists at which, for a specific cell (i.e., a specific permeability and cell size), the cellular dehydration is ideally compensated by

## *The Use of Chitooligosaccharides in Cryopreservation: Discussion of Concept and First… DOI: http://dx.doi.org/10.5772/intechopen.89162*

the CPA penetration. This rate deals with the two major risks of the slow-freezing procedures, the "solute effect," which occurs at low cooling rate, and the intracellular nucleation, which occurs at high cooling rate [101].

The addition of penetrating CPA in a solution, in a reasonable proportion (> 1 mol. L<sup>1</sup> for slow-freezing and > 3 mol.L<sup>1</sup> for vitrification), is considered essential for the survival of mammalian embryo type cells [21]. Very few studies have been published on attempts to reduce the amount of Me2SO in solution during slow-freezing cryopreservation [43, 84]. This strategy was evaluated by relying on modeling arguments, showing that it seems complicated to reduce this initial proportion of penetrating CPA [21]. We make a proposal for slow-freezing procedure optimization using a reduced initial proportion of penetrating CPA, supplemented with non-penetrating CPA.

The decrease of the initial proportion of penetrating CPA implies an increase in the amount of crystallizable water, a stronger evolution of the crystal/liquid ratio in the solution with temperature, a higher amount of penetrating CPA needed to diffuse through the membrane, more risks of contact between cells and ice crystals, etc. Thus, penetrating CPA play a cryoprotective role that cannot be fully reproduced in the extracellular environment by non-penetrating CPA. Moreover, the stresses and risks associated with the ice formation must be limited or prevented until the plunge to LN2. It is therefore necessary to continue to use penetrating CPA, but their amount in the initial solution should be limited to reduce cytotoxicity. In addition to the presence of penetrating CPA, the vitrification of the extracellular remaining solution depends on the presence of non-penetrating CPA that limit the evolution of penetrating CPA concentrations and reduce the risks associated with the ice formation in solution. The reduction of penetrating CPA implies to control the damages related to the ice formation in solution by replacing a large part of the initial extracellular water by non-crystallizable substances, which offer favorable conditions for cryopreservation. However, the biocompatibility of these compounds on the cellular systems must be guaranteed.

Because the proportion of CPA in the intracellular medium has a lighter impact than the cellular dehydration on the intracellular vitrification [16, 87, 94–96], we assume that the proportion of penetrating CPA may be limited. In that case, it is then necessary to reduce the deleterious effects related to the cellular dehydration by using non-penetrating CPA that are not highly osmotically active and whose molar concentrations slightly change during cooling (i.e., compounds with a large molar mass). It is also necessary to employ the "fast interrupt protocol" by suitably combining dehydration and lowering temperature so that the colloidal intracellular transition occurs before cell dehydration becomes too important.

Finally, it is necessary to verify that the extracellular remaining solution can vitrify easily so that the cryopreservation procedure can be interrupted at a higher plunge temperature into LN2. Thus, the use of cryostabilizer CPA that facilitate the achievement of the vitreous state, without binding a significant amount of water, may promote the stabilization of the system "ice/remaining solution" (by reducing the risk of recrystallization of the intercrystalline remaining solution).

We chose to study the possibility to cryopreserve mouse embryos with 5% (v/v) of Me2SO in the initial solution, supplemented with non-penetrating CPA (COS).

## **4. The proposition to use COS**

The idea of using COS compounds in cell cryopreservation is supported by:

• An absence of toxicity, an ability to degrade without toxic residues, and a biocompatibility [102].

cell cytoplasm is often heavily loaded with large molecules, it can easily vitrify [17, 59, 96]). In a vitrification procedure, using successive baths of osmotic equilibration, the cells are placed in solutions containing penetrating CPA. It was proposed by Kuleshova et al. [17] to replace, in those procedures, a large proportion of penetrating CPA with non-penetrating polymers (PVP, dextran, Ficoll, etc.). In the last osmotic equilibration bath, non-penetrating CPA (sugars or polymers) are added to promote cell dehydration just before the plunge into liquid nitrogen (LN2). The osmotic effect of these non-penetrating CPA promotes the vitrification of the intracellular medium and ensures the success of the procedure with a lower amount of penetrating CPA [16]. By adaptation to slow-freezing procedures, we assume that it is possible to force the intracellular colloidal glass transition to the correct temperature by suitably combining temperature lowering and cellular dehydration. However, cellular dehydration is not possible over long periods when the temperature remains high. During a vitrification procedure, the dehydration time is short (primarily applied during the last osmotic bath, a few seconds before the plunge into LN2). During slow-freezing, if the dehydration intervenes too extremely, it could lead to cellular collapse [83, 98, 99]. To solve this problem, we rely on the work of Mazur et al. [100] who proposed an alternative to conventional cellular cryopreservation protocols by rapidly bringing the cells to a temperature

*Cryopreservation - Current Advances and Evaluations*

low enough to make the effects of cell dehydration less deleterious. This temperature must, however, remain above the nucleation temperature of the intracellular medium. A temperature stabilization is then carried out at this

the extracellular media.

**8**

**3.2 The elements of the proposed optimization**

intermediate temperature to allow the equilibration of the media. Then, the cooling continues at a slow cooling rate until the plunge into LN2. This type of protocol is called "rapid cooling interrupted" [100]. It should be noted that this protocol also possesses a seeding step in order to control the extracellular ice growth. We assume that, when using this type of protocol, it would be possible to allow faster cell dehydration already biologically tolerated in the case of vitrification procedures.

c. The use of non-penetrating CPA can allow reducing the necessary proportion of penetrating CPA by optimizing the procedure of cooling and warming. For a slow-freezing procedure, in which ice formation is allowed and desired, the osmotic effect of a non-penetrating CPA becomes increasingly important as the proportion of water in the remaining extracellular medium decreases. Conversely, in the intracellular medium, it is mainly the penetrating CPA that participates in the osmotic balance. There is therefore a gap between these two media in the osmolality evolution, which accentuates cellular dehydration and increases cell contraction at a given cooling rate. The use of an osmotically active non-penetrating CPA in solution may induce a significant change in the osmolality difference between the intra- and

The toxicity of the cryopreservation solutions is related to the CPA concentration

reached in the remaining solution to enable the vitrification of the intracellular medium. Moreover, to protect the cells, the vitrification must also be carried out in the noncrystalline extracellular medium in contact with the cells. Intracellular vitrification is dependent on the presence of penetrating CPA and on the cellular dehydration. In consequence, the intracellular vitrification is dependent on the diffusion of materials across the cellular membrane. That is why cryopreservation procedures are time dependent. Indeed, an ideal cooling rate exists at which, for a specific cell (i.e., a specific permeability and cell size), the cellular dehydration is ideally compensated by


thermogravimetric analysis has shown that there are residual hydration levels after synthesis, purification, and lyophilization, which are variable according to the DA parameter of the chitosan polymers chains. From this data, we extrapolated the hydration levels of the COS compounds according to the DA parameter. For a DA ≈ 0%, we considered a residual hydration value of the COS compound equal to 6.5% (w/w). This is consistent with other results published in the literature where a

*The Use of Chitooligosaccharides in Cryopreservation: Discussion of Concept and First…*

Before considering the use of COS in cryopreservation solutions, an evaluation of its biocompatibility is necessary. It ensures that their presence will not be deleterious to the future survival and development of the cryopreserved biological system. By their nature and chemical composition, the biocompatibility of COS has already been emphasized [113]. However, the molecular interactions between all the compounds in extracellular solution and the cell membranes are complex. There are, for example, interactions between COS molecules and cell membranes that are invoked to explain their fungistatic and bacteriostatic properties [113]. It is therefore not excluded that interactions may exist with eukaryotic cells. In addition, the cell system chosen for this study, the mouse embryo, is a fragile system whose development can be disrupted by the presence of harmful materials. Consequently, deleterious influence on this cellular system, linked to COS, must be excluded.

The biocompatibility of COS was evaluated with mouse embryos. A procedure identical to that presented in a previous team article [109] was applied for the production of embryos used in this study. To study the biological action of COS on

**embryos in culture**

1 IMV 7.29 25 23 92% 23 92% 23 92% 2 IMV + 10% (v/v) Me2SO 7.29 25 20 80% 18 72% 14 56% 3 IMV + 5% (v/v) Me2SO 7.30 25 23 92% 20 80% 20 80%

*The stage reached by the embryos was evaluated under a binocular microscope every 24 hours for 4 days. For each embryonic stage, the number of embryos observed at this stage is indicated in the left column and, on the right, the*

**Young blastocysts**

7.32 25 25 100% 22 88% 20 80%

) COS10\_0 7.34 26 24 92% 23 88% 23 88%

cryopreservation solution (IMV + 5% (v/v) Me2SO) and in the "IMV" holding medium. One hundred twenty-six embryos (morula stage) were collected and were then mixed and divided into five groups (cf. **Table 2**): (1) "IMV," a control group placed in a solution without CPA; (2) "IMV + 10% (v/v) Me2SO," a control group placed in the solution conventionally used for cryopreservation of mouse embryos; (3) "IMV + 5% (v/v) Me2SO," a control group placed in the solution in which COS

) was dissolved in the

**Expanded blastocyst** **Hatching**

mouse embryos, a large quantity of COS10\_0 (150 mg.mL<sup>1</sup>

**Groups pH Number of**

value of 6% (w/w) was obtained [112].

*DOI: http://dx.doi.org/10.5772/intechopen.89162*

**6.1 Materials and methods**

4 IMV + 5% (v/v) Me2SO + (150 mg.mL<sup>1</sup>

5 IMV + (150 mg.mL<sup>1</sup>

**Table 2.**

**11**

) COS10\_0

*Compilation of embryonic developments observed by groups.*

*corresponding percentage in relation to the initial number of cultured embryos.*

**6. Questioning the biocompatibility of COS**

• Potential interactions with water and consequently a potential effect on the properties of aqueous solutions (like that proposed by mono-, di-, and oligosaccharides).

In addition, because of the length of their chains, the COS propose intermediate properties between the polymers and the mono- or disaccharides. These properties are adjustable since the length of the chains can be selected during their synthesis using degree of polymerization (DP) parameter as well as the nature of the monomers present on the chain using the degree of acetylation (DA) parameter. These modular characteristics let us hope for the attainment of physicochemical properties that are adjustable for use as a CPA. Furthermore, they are particularly biocompatible and nontoxic. As a result, addition of COS to some penetrating CPA appears to be a relevant choice to reduce the needed amount of CPA during slow-freezing.

Assuming that a use of COS would reduce the use of Me2SO, this work proposes to look for a composition of COS-based solution that can reproduce, in the extracellular medium, some effects of Me2SO (favor the vitrification of the intracellular medium, promote the vitrification of the intercrystalline remaining solution, reduce the cell dehydration, etc.).

Our current procedure used for mouse embryo cryopreservation has been optimized for the "IMV" holding medium (embryo-holding medium, IMV® Technologies, L'Aigle, France) supplemented with approximately 10% (v/v) Me2SO [109]. We set the goal of reducing by 50% the proportion of Me2SO, to successfully cryopreserve mouse embryos using an IMV + 5% (v/v) Me2SO solution containing a certain amount of COS.

## **5. Synthesis and chemical characterization of COS**

The basic compound used to synthesize COS is chitosan supplied by the Indian company Mahtani Chitosan®. This chitosan (batch 244/020208) is produced from chitin extracted from shrimp shells. The chitosan provided was almost completely deacetylated (DA < 0.5%) by deacetylation reaction. This chitosan has a numberaverage molecular weight of 115 kg.mol<sup>1</sup> and dispersity of 2.3. For the preparation of COS, the macromolecular chitosan chains are depolymerized according to a nitrous acid deamination reaction using sodium nitrite (NaNO2) in acidic conditions [110]. It is only possible to obtain a statistical average chain length distribution around a mean DP value. To control the DA parameter, it is possible to perform an N-acetylation reaction of the D-glucosamine units with acetic anhydride as acetylation agent. In this chapter, DP and DA parameters have been used to name the COS as follows: COSDP\_DA, with DP = "the average number of monomers per COS chain" and DA = "the average degree of acetylation of the chains of this COS."

To estimate the residual hydration rate in purified COS, we used data from the literature. A previous study of the same types of chitosan chains, but in a polymeric form, evaluated these hydration levels [111]. This study performed by

*The Use of Chitooligosaccharides in Cryopreservation: Discussion of Concept and First… DOI: http://dx.doi.org/10.5772/intechopen.89162*

thermogravimetric analysis has shown that there are residual hydration levels after synthesis, purification, and lyophilization, which are variable according to the DA parameter of the chitosan polymers chains. From this data, we extrapolated the hydration levels of the COS compounds according to the DA parameter. For a DA ≈ 0%, we considered a residual hydration value of the COS compound equal to 6.5% (w/w). This is consistent with other results published in the literature where a value of 6% (w/w) was obtained [112].

## **6. Questioning the biocompatibility of COS**

Before considering the use of COS in cryopreservation solutions, an evaluation of its biocompatibility is necessary. It ensures that their presence will not be deleterious to the future survival and development of the cryopreserved biological system. By their nature and chemical composition, the biocompatibility of COS has already been emphasized [113]. However, the molecular interactions between all the compounds in extracellular solution and the cell membranes are complex. There are, for example, interactions between COS molecules and cell membranes that are invoked to explain their fungistatic and bacteriostatic properties [113]. It is therefore not excluded that interactions may exist with eukaryotic cells. In addition, the cell system chosen for this study, the mouse embryo, is a fragile system whose development can be disrupted by the presence of harmful materials. Consequently, deleterious influence on this cellular system, linked to COS, must be excluded.

## **6.1 Materials and methods**

• Bacteriological, fungistatic, and antitumor properties [102–106].

• A tendency to form aggregates in aqueous media [108].

*Cryopreservation - Current Advances and Evaluations*

in cryobiology.

oligosaccharides).

the cell dehydration, etc.).

certain amount of COS.

**10**

• An ability to be soluble in aqueous solutions at physiological pH [107].

• A chemical structure of oligosaccharide type close to molecules currently used

• Potential interactions with water and consequently a potential effect on the properties of aqueous solutions (like that proposed by mono-, di-, and

In addition, because of the length of their chains, the COS propose intermediate properties between the polymers and the mono- or disaccharides. These properties are adjustable since the length of the chains can be selected during their synthesis using degree of polymerization (DP) parameter as well as the nature of the monomers present on the chain using the degree of acetylation (DA) parameter. These modular characteristics let us hope for the attainment of physicochemical properties that are adjustable for use as a CPA. Furthermore, they are particularly biocompatible and nontoxic. As a result, addition of COS to some penetrating CPA appears to be a relevant choice to reduce the needed amount of CPA during slow-freezing. Assuming that a use of COS would reduce the use of Me2SO, this work proposes to look for a composition of COS-based solution that can reproduce, in the extracellular medium, some effects of Me2SO (favor the vitrification of the intracellular medium, promote the vitrification of the intercrystalline remaining solution, reduce

Our current procedure used for mouse embryo cryopreservation has been optimized for the "IMV" holding medium (embryo-holding medium, IMV® Technologies, L'Aigle, France) supplemented with approximately 10% (v/v) Me2SO [109]. We set the goal of reducing by 50% the proportion of Me2SO, to successfully cryopreserve mouse embryos using an IMV + 5% (v/v) Me2SO solution containing a

The basic compound used to synthesize COS is chitosan supplied by the Indian company Mahtani Chitosan®. This chitosan (batch 244/020208) is produced from chitin extracted from shrimp shells. The chitosan provided was almost completely deacetylated (DA < 0.5%) by deacetylation reaction. This chitosan has a numberaverage molecular weight of 115 kg.mol<sup>1</sup> and dispersity of 2.3. For the preparation of COS, the macromolecular chitosan chains are depolymerized according to a nitrous acid deamination reaction using sodium nitrite (NaNO2) in acidic conditions [110]. It is only possible to obtain a statistical average chain length distribution around a mean DP value. To control the DA parameter, it is possible to perform an N-acetylation reaction of the D-glucosamine units with acetic anhydride as acetylation agent. In this chapter, DP and DA parameters have been used to name the COS as follows: COSDP\_DA, with DP = "the average number of monomers per COS chain" and DA = "the average degree of acetylation of the chains of this COS." To estimate the residual hydration rate in purified COS, we used data from the literature. A previous study of the same types of chitosan chains, but in a polymeric

**5. Synthesis and chemical characterization of COS**

form, evaluated these hydration levels [111]. This study performed by

The biocompatibility of COS was evaluated with mouse embryos. A procedure identical to that presented in a previous team article [109] was applied for the production of embryos used in this study. To study the biological action of COS on mouse embryos, a large quantity of COS10\_0 (150 mg.mL<sup>1</sup> ) was dissolved in the cryopreservation solution (IMV + 5% (v/v) Me2SO) and in the "IMV" holding medium. One hundred twenty-six embryos (morula stage) were collected and were then mixed and divided into five groups (cf. **Table 2**): (1) "IMV," a control group placed in a solution without CPA; (2) "IMV + 10% (v/v) Me2SO," a control group placed in the solution conventionally used for cryopreservation of mouse embryos; (3) "IMV + 5% (v/v) Me2SO," a control group placed in the solution in which COS


*The stage reached by the embryos was evaluated under a binocular microscope every 24 hours for 4 days. For each embryonic stage, the number of embryos observed at this stage is indicated in the left column and, on the right, the corresponding percentage in relation to the initial number of cultured embryos.*

## **Table 2.**

*Compilation of embryonic developments observed by groups.*

are diluted; (4) "IMV + 5% (v/v) Me2SO + (150 mg.mL<sup>1</sup> ) COS10\_0," a test group placed in a tested cryopreservation solution with COS; and (5) "IMV + (150 mg. mL<sup>1</sup> ) COS10\_0," a second test group, without Me2SO, studied to highlight the effect of COS and avoid the potentially cross-cutting effects between COS and Me2SO.

thermodynamic characterization of polymer chains. To our knowledge, the characterization of the thermodynamic properties of aqueous solutions formulated with soluble oligosaccharide derivatives of chitin or chitosan has not yet been done. The objective of this study is to evaluate the impact of COS on the thermodynamic properties of aqueous solutions and to put forward a potential action of these products on water and other properties of the solutions (viscosity, gelling, etc.). It

*The Use of Chitooligosaccharides in Cryopreservation: Discussion of Concept and First…*

A differential scanning calorimeter (DSC Diamond; Perkin Elmer) with power compensation was used in this study. The previously described procedures [117] have been applied, with the same methods of sample preparation, calibration of the DSC calorimeter, and precautions for use. COS were added in the IMV + 5% (v/v)

of COS (per unit volume of the IMV + 5% (v/v) Me2SO solution), as listed in

protocol starts with a cooling from +10°C to �150°C at �100°C.min�<sup>1</sup>

, and a warming from �150°C to +20°C at +20°C.min�<sup>1</sup>

were repeated three times (n = 3) for each concentration.

*7.1.2 Correction of the studied mass concentrations*

product. Correction is made according to Eq. (1):

Real mass concentration of COS (mg.mL�<sup>1</sup>

**Averaged DA (%)**

lyophilized powder of purified COS (Ø)

*purification) and their studied mass concentrations.*

of hydrated COS (mg.mL�<sup>1</sup>

**DP**

**Products Averaged**

**Table 3.**

**13**

To study the cryoprotective capability of COS as a substitute for Me2SO, the solutions were studied by DSC using a protocol described elsewhere [117]. This

The studied COS have a residual moisture content. Thus, the mass concentration of the added powder is not equivalent to the real mass concentration of the added

*Real mass concentration of COS* ¼ *Powder mass concentration of hydrated COS*

**Residual hydration ratio after purification % (w/w)**

COS7.5\_0 7.5 0 6.5 30; 60; 90; 150 28.05; 56.1;

*Table of the studied products, with their characteristics (DP, DA, and residual hydration ratio after*

COS10\_0 10 0 6.5 0; 25; 50; 100;

<sup>∗</sup> <sup>1</sup> � <sup>Ψ</sup>H2O*,*inið Þ COS

), ΨH2O*,*inið Þ COS , residual hydration ratio in the

**Powder mass concentrations studied (mg.mL**�**<sup>1</sup> )**

150; 200

) of hydrated powder

. The experiments

, a cooling from +10°C to �150°C at �2.5°C.

), powder mass concentration

**Real mass concentrations studied (mg.mL**�**<sup>1</sup> )**

84.15; 140.3

0; 23.38; 46.75; 93.5; 140.3; 187

, a warming

(1)

**DSC sample mass (mg)**

�5

�10

will help to evaluate the cryoprotection effect of COS.

Me2SO solution, with different mass concentrations (mg.mL�<sup>1</sup>

*7.1.1 Thermodynamic characterization of solutions*

*DOI: http://dx.doi.org/10.5772/intechopen.89162*

from �150°C to +10°C at +2.5°C.min�<sup>1</sup>

**7.1 Materials and methods**

**Table 3**.

min�<sup>1</sup>

The embryos were placed in these solutions at room temperature for 10 minutes. They were rinsed with M16 culture medium (IMV® Technologies, L'Aigle, France) and then placed in a culture chamber. The culture medium was equilibrated in the incubator (+37°C; 5% (v/v) CO2; humid atmosphere), and then the embryos were introduced therein. The culture was maintained and supervised for 4 days. The development of embryos was then compared (every 24 hours) with other control groups to assess the state of the embryonic stage reached by each embryo. These comparisons can highlight certain harmful effects related to the presence of the different products on cellular development.

## **6.2 Results and discussions**

After 24 hours, 20% of the embryos in the 10% (v/v) Me2SO group failed to reach the "young blastocyst" stage. For groups with 5% (v/v) Me2SO, the development stage remains similar to the solution without Me2SO. The presence of COS does not appear to have a deleterious effect on the development of embryos at this stage, and the reduction in the proportion of Me2SO in solution seems to be beneficial.

After 48 hours, it is possible to compare the number of embryos which reached the "expanded blastocyst" stage to the number of embryos which previously reached the "young blastocyst" stage. The numbers are the same for group 1 (control without CPA and COS), while an embryo did not develop in group 5 (with COS but without CPA), and more than one embryo did not develop in the other groups (2, 3, and 4). By comparing the results for groups 3 and 4, the effect of COS presence on embryo development may be considered minimal. Conversely, the decrease in the number of living embryos in groups 2, 3, and 4 seems to be directly associated with the presence of Me2SO in the solution where embryos are bathed at room temperature.

After 72 hours, the number of embryos that reached the hatching stage is the least important for group 2 (with 10% (v/v) Me2SO). There is no difference according to the presence or absence of COS in the IMV, and the final percentage in each case is very close. It is the same in solutions with 5% (v/v) Me2SO, where the number of embryos which reached this stage of development is equivalent, with or without COS.

According to this study repeated only once, a negative effect of Me2SO on the development of mouse embryos is highlighted. However, a negative effect of COS on this cellular development is discarded. These results should be repeated but seem to demonstrate the biocompatibility of COS for the mouse embryos. We conclude that COS can be used in the cryopreservation solutions in contact with cells.

## **7. Thermodynamic characterization of cryopreservation solutions containing COS**

The strategy outlined in Part 3 aims to reduce by 50% the initial volume proportion of Me2SO in slow-freezing solutions with the help of COS. It is thus necessary to know more precisely their mode of action in aqueous solutions. Several studies have already proposed the thermodynamic characterization of chitosan or chitosan derivatives [26, 112, 114–116]. However, these studies have focused on the

## *The Use of Chitooligosaccharides in Cryopreservation: Discussion of Concept and First… DOI: http://dx.doi.org/10.5772/intechopen.89162*

thermodynamic characterization of polymer chains. To our knowledge, the characterization of the thermodynamic properties of aqueous solutions formulated with soluble oligosaccharide derivatives of chitin or chitosan has not yet been done.

The objective of this study is to evaluate the impact of COS on the thermodynamic properties of aqueous solutions and to put forward a potential action of these products on water and other properties of the solutions (viscosity, gelling, etc.). It will help to evaluate the cryoprotection effect of COS.

## **7.1 Materials and methods**

are diluted; (4) "IMV + 5% (v/v) Me2SO + (150 mg.mL<sup>1</sup>

*Cryopreservation - Current Advances and Evaluations*

different products on cellular development.

**6.2 Results and discussions**

room temperature.

without COS.

**containing COS**

**12**

mL<sup>1</sup>

Me2SO.

placed in a tested cryopreservation solution with COS; and (5) "IMV + (150 mg.

) COS10\_0," a second test group, without Me2SO, studied to highlight the effect of COS and avoid the potentially cross-cutting effects between COS and

The embryos were placed in these solutions at room temperature for 10 minutes. They were rinsed with M16 culture medium (IMV® Technologies, L'Aigle, France) and then placed in a culture chamber. The culture medium was equilibrated in the incubator (+37°C; 5% (v/v) CO2; humid atmosphere), and then the embryos were introduced therein. The culture was maintained and supervised for 4 days. The development of embryos was then compared (every 24 hours) with other control groups to assess the state of the embryonic stage reached by each embryo. These comparisons can highlight certain harmful effects related to the presence of the

After 24 hours, 20% of the embryos in the 10% (v/v) Me2SO group failed to reach the "young blastocyst" stage. For groups with 5% (v/v) Me2SO, the development stage remains similar to the solution without Me2SO. The presence of COS does not appear to have a deleterious effect on the development of embryos at this stage, and

After 48 hours, it is possible to compare the number of embryos which reached

After 72 hours, the number of embryos that reached the hatching stage is the

According to this study repeated only once, a negative effect of Me2SO on the development of mouse embryos is highlighted. However, a negative effect of COS on this cellular development is discarded. These results should be repeated but seem to demonstrate the biocompatibility of COS for the mouse embryos. We conclude that COS can be used in the cryopreservation solutions in contact with cells.

least important for group 2 (with 10% (v/v) Me2SO). There is no difference according to the presence or absence of COS in the IMV, and the final percentage in each case is very close. It is the same in solutions with 5% (v/v) Me2SO, where the number of embryos which reached this stage of development is equivalent, with or

**7. Thermodynamic characterization of cryopreservation solutions**

The strategy outlined in Part 3 aims to reduce by 50% the initial volume proportion of Me2SO in slow-freezing solutions with the help of COS. It is thus necessary to know more precisely their mode of action in aqueous solutions. Several studies have already proposed the thermodynamic characterization of chitosan or chitosan derivatives [26, 112, 114–116]. However, these studies have focused on the

the reduction in the proportion of Me2SO in solution seems to be beneficial.

the "expanded blastocyst" stage to the number of embryos which previously reached the "young blastocyst" stage. The numbers are the same for group 1 (control without CPA and COS), while an embryo did not develop in group 5 (with COS but without CPA), and more than one embryo did not develop in the other groups (2, 3, and 4). By comparing the results for groups 3 and 4, the effect of COS presence on embryo development may be considered minimal. Conversely, the decrease in the number of living embryos in groups 2, 3, and 4 seems to be directly associated with the presence of Me2SO in the solution where embryos are bathed at

) COS10\_0," a test group

## *7.1.1 Thermodynamic characterization of solutions*

A differential scanning calorimeter (DSC Diamond; Perkin Elmer) with power compensation was used in this study. The previously described procedures [117] have been applied, with the same methods of sample preparation, calibration of the DSC calorimeter, and precautions for use. COS were added in the IMV + 5% (v/v) Me2SO solution, with different mass concentrations (mg.mL�<sup>1</sup> ) of hydrated powder of COS (per unit volume of the IMV + 5% (v/v) Me2SO solution), as listed in **Table 3**.

To study the cryoprotective capability of COS as a substitute for Me2SO, the solutions were studied by DSC using a protocol described elsewhere [117]. This protocol starts with a cooling from +10°C to �150°C at �100°C.min�<sup>1</sup> , a warming from �150°C to +10°C at +2.5°C.min�<sup>1</sup> , a cooling from +10°C to �150°C at �2.5°C. min�<sup>1</sup> , and a warming from �150°C to +20°C at +20°C.min�<sup>1</sup> . The experiments were repeated three times (n = 3) for each concentration.

## *7.1.2 Correction of the studied mass concentrations*

The studied COS have a residual moisture content. Thus, the mass concentration of the added powder is not equivalent to the real mass concentration of the added product. Correction is made according to Eq. (1):

$$\begin{aligned} \text{Real mass concentration of COS} &= \text{Power mass concentration of hydrates COS} \\ &\approx \left(1 - \Psi\_{\text{H}\_2\text{O}\_3\text{ini}}(\text{COS})\right) \end{aligned} \tag{1}$$

Real mass concentration of COS (mg.mL�<sup>1</sup> ), powder mass concentration of hydrated COS (mg.mL�<sup>1</sup> ), ΨH2O*,*inið Þ COS , residual hydration ratio in the lyophilized powder of purified COS (Ø)


**Table 3.**

*Table of the studied products, with their characteristics (DP, DA, and residual hydration ratio after purification) and their studied mass concentrations.*

The actual mass concentrations of COS in the solutions that were studied are presented in **Table 3**. In the following analyses, only the actual mass concentration of COS is considered for a given compound. However, to simplify the notation in thermograms, the hydrated powdered mass concentration of COS is used to designate the solutions.

lower than the theoretical ones materialize an evolution of the proportion of solution which is crystallized. Indeed, it materializes a deviation in the evolution of

*The Use of Chitooligosaccharides in Cryopreservation: Discussion of Concept and First…*

the compound concentrations within the remaining solution (according to

transition of liquid water to ice for an IMV + 5% (v/v) Me2SO solution with a

ΔHcalculated with an inert massð Þ¼ ½ � c ΔHmeasuredð Þ ½ �¼ c 0 ∗ ΨIMVþ5% vð Þ *<sup>=</sup>*<sup>v</sup> Me2SO (3)

where ΔHcalculated with an inert mass([c]) is the calculated specific enthalpy for the

), and ΨIMVþ5% vð Þ *<sup>=</sup>*<sup>v</sup> Me2SO is the initial mass proportion of the

measured specific enthalpy for the transition of liquid water to ice for a IMV + 5% (v/v) Me2SO solution = 265.68 � 3.2 J.g�<sup>1</sup> (this specific enthalpy value corresponds to the average of the three measurements with the IMV + 5% (v/v) Me2SO solution

IMV + 5% (v/v) Me2SO solution in the prepared IMV + 5% (v/v) Me2SO solution

Eq. (4) allows estimating the initial mass proportion of the IMV + 5% (v/v) Me2SO solution in the prepared IMV + 5% (v/v) Me2SO solution containing an inert

mIMVþ5%Me2SO þ mproduct

where ΨIMVþ5% vð Þ *<sup>=</sup>*<sup>v</sup> Me2SO is the initial mass proportion of the IMV + 5% (v/v) Me2SO solution in the prepared IMV + 5% (v/v) Me2SO solution containing an inert

the volume of the solution (mL), and [product] is the initial mass concentration of

A cryoscopic depression indicates the action of a dissolved product on the properties of water molecules in solution, providing information on interactions that occur between this product and the water molecules. The estimation of the maximal equilibrium temperature (Tm), for different concentrations of the same COS introduced in the IMV + 5% (v/v) Me2SO solution, informs about the cryoscopic depression induced by the oligosaccharide. Moreover, knowing the temperature (Tm) of a solution allows to estimate, as a function of the temperature, the

Due to the kinetic phenomena of heat transfer, it is often imprecise to confuse the temperature recorded at the maximum of the endothermic melting peak (Tmax) with the maximum melting point temperature of the ice in solution, because Tm is less or equal to Tmax [119]. In the kinetic phenomena being influenced by the mass involved in the melting process, the comparison of the Tmax values becomes difficult between the series for which the mass of sample studied is different and in which the mass of water in solution concerned by the melting is different.

supercooling magnitude reached in this mixture (before crystallization).

).

<sup>¼</sup> <sup>1014</sup>*:*<sup>5</sup>

mass (Ø), dIMV + 5%Me2SO is the density of the IMV + 5%(v/v) Me2SO solution = 1.0145 (experimentally measured with a pipette and a high sensibility weighing scale) (Ø), ρH2O is the mass volume of water ≈ 1000 (mg.mL�<sup>1</sup>

<sup>¼</sup> dIMVþ5%Me2SO <sup>∗</sup> <sup>ρ</sup>H2O <sup>∗</sup> Vsol

dIMVþ5%Me2SO ∗ ρH2O ∗ Vsol þ ½ � product ∗ Vsol

<sup>1014</sup>*:*<sup>5</sup> <sup>þ</sup> ½ � product (4)

), Vsol is

), ΔHmeasured([c] = 0) is the experimentally

the lever rule).

without COS) (J.g�<sup>1</sup>

mass such as COS.

**15**

containing an inert mass (Ø).

concentration [c] of inert mass (J.g�<sup>1</sup>

*DOI: http://dx.doi.org/10.5772/intechopen.89162*

<sup>Ψ</sup>IMVþ5% vð Þ *<sup>=</sup>*<sup>v</sup> Me2SOð Þ¼ sol mIMVþ5%Me2SO

hydrated product added in solution (mg.mL�<sup>1</sup>

*7.1.5 Estimation of the cryoscopic depression*

## *7.1.3 Normalization of the transition enthalpies to 0°C*

The amounts of ice formed in solution were estimated using the determination of the crystallization enthalpy (ΔHc) and the melting enthalpy (ΔHm). To estimate these amounts, the peak areas were calculated using a sigmoidal curve baseline (with the Pyris software 11.1.1). During warming the tangents, allowing the calculation of the sigmoidal line, were positioning, respectively, just before the colloidal transition and just after the melting peak.

In order to compare the transition enthalpies, a normalization of the measured values to a specified temperature of 0°C was made. As the enthalpy value of the transition of water to ice evolves as a function of temperature, the "latent heat of solidification of supercooled water" (Lf) was obtained at the measured phase change temperature from Boutron's [118] data interpolation. His data were calculated [119] from Angell's [120] specific heat capacity (Cp) of supercooled water and from Weast's Hand Book's [121] Cp of ice at different temperatures.

The normalization of the measured values to the expected values at the specified temperature of 0°C considers that the same proportion of solution transits at the transition temperature (Tt) (ΔHmeas(Tt)/Lf(Tt)) and at 0°C (ΔHnormalized(0°C)/ Lf(0°C)). This equality leads to Eq. (2):

$$
\Delta \mathbf{H}\_{\text{normalized}}(\mathbf{0}^{\bullet}\mathbf{C}) = \frac{\Delta \mathbf{H}\_{\text{meas}}(\mathbf{T}\_{\text{t}}) \* \mathbf{L}\_{\text{f}}(\mathbf{0}^{\bullet}\mathbf{C})}{\mathbf{L}\_{\text{f}}(\mathbf{T}\_{\text{t}})} \tag{2}
$$

where ΔHnormalized(0°C) is the transition enthalpy of the sample normalized to 0°C (J.g�<sup>1</sup> ), ΔHmeas(Tt) is the transition enthalpy of the sample measured at a transition temperature Tt (J.g�<sup>1</sup> ), Lf(Tt) is the transition enthalpy of a pure water sample measured at a transition temperature Tt (J.g�<sup>1</sup> ), and Lf(0°C) is the transition enthalpy of a pure water sample measured at 0°C (= 333.4 J.g�<sup>1</sup> ) (J.g�<sup>1</sup> ).

During cooling, to analyze the crystallization enthalpies, the nucleation temperature (Tn) was considered as the transition temperature (i.e., Tt = Tn). During warming, to analyze the melting enthalpies, the temperature of the summit of the melting peak (Tmax) was considered as the transition temperature (i.e., Tt = Tmax). These are approximations because for a mixture there is not only one transition temperature.

## *7.1.4 Comparison of measured and calculated transition enthalpies with the addition of an inert mass*

When a non-hydrated inert mass is added in a solution, a lowering of the enthalpies of crystallization and melting is expected since this addition decreases, in proportion, the mass of water present in solution. The term "inert" refers to a product that does not influence the transition enthalpies (i.e., a product that does not transit and does not influence the transition of other products). The theoretical link between the decrease of the transition enthalpies and the decrease of the mass proportion of water in solution is given by Eq. (3). The comparison of a theoretical value with a measured one allows estimating the mass of water which transits in comparison to the mass of water present within the solution. Experimental values

lower than the theoretical ones materialize an evolution of the proportion of solution which is crystallized. Indeed, it materializes a deviation in the evolution of the compound concentrations within the remaining solution (according to the lever rule).

$$
\Delta \mathbf{H}\_{\text{calculated with an inert mass}}([\mathbf{c}]) = \Delta \mathbf{H}\_{\text{measured}}([\mathbf{c}] = \mathbf{0}) \ast \Psi\_{\text{IMV} + \\$\mathbb{W}(\mathbf{v}/\mathbf{v})\text{Me}\_2\text{SO}}\tag{3}
$$

where ΔHcalculated with an inert mass([c]) is the calculated specific enthalpy for the transition of liquid water to ice for an IMV + 5% (v/v) Me2SO solution with a concentration [c] of inert mass (J.g�<sup>1</sup> ), ΔHmeasured([c] = 0) is the experimentally measured specific enthalpy for the transition of liquid water to ice for a IMV + 5% (v/v) Me2SO solution = 265.68 � 3.2 J.g�<sup>1</sup> (this specific enthalpy value corresponds to the average of the three measurements with the IMV + 5% (v/v) Me2SO solution without COS) (J.g�<sup>1</sup> ), and ΨIMVþ5% vð Þ *<sup>=</sup>*<sup>v</sup> Me2SO is the initial mass proportion of the IMV + 5% (v/v) Me2SO solution in the prepared IMV + 5% (v/v) Me2SO solution containing an inert mass (Ø).

Eq. (4) allows estimating the initial mass proportion of the IMV + 5% (v/v) Me2SO solution in the prepared IMV + 5% (v/v) Me2SO solution containing an inert mass such as COS.

<sup>Ψ</sup>IMVþ5% vð Þ *<sup>=</sup>*<sup>v</sup> Me2SOð Þ¼ sol mIMVþ5%Me2SO mIMVþ5%Me2SO þ mproduct <sup>¼</sup> dIMVþ5%Me2SO <sup>∗</sup> <sup>ρ</sup>H2O <sup>∗</sup> Vsol dIMVþ5%Me2SO ∗ ρH2O ∗ Vsol þ ½ � product ∗ Vsol <sup>¼</sup> <sup>1014</sup>*:*<sup>5</sup> <sup>1014</sup>*:*<sup>5</sup> <sup>þ</sup> ½ � product (4)

where ΨIMVþ5% vð Þ *<sup>=</sup>*<sup>v</sup> Me2SO is the initial mass proportion of the IMV + 5% (v/v) Me2SO solution in the prepared IMV + 5% (v/v) Me2SO solution containing an inert mass (Ø), dIMV + 5%Me2SO is the density of the IMV + 5%(v/v) Me2SO solution = 1.0145 (experimentally measured with a pipette and a high sensibility weighing scale) (Ø), ρH2O is the mass volume of water ≈ 1000 (mg.mL�<sup>1</sup> ), Vsol is the volume of the solution (mL), and [product] is the initial mass concentration of hydrated product added in solution (mg.mL�<sup>1</sup> ).

## *7.1.5 Estimation of the cryoscopic depression*

A cryoscopic depression indicates the action of a dissolved product on the properties of water molecules in solution, providing information on interactions that occur between this product and the water molecules. The estimation of the maximal equilibrium temperature (Tm), for different concentrations of the same COS introduced in the IMV + 5% (v/v) Me2SO solution, informs about the cryoscopic depression induced by the oligosaccharide. Moreover, knowing the temperature (Tm) of a solution allows to estimate, as a function of the temperature, the supercooling magnitude reached in this mixture (before crystallization).

Due to the kinetic phenomena of heat transfer, it is often imprecise to confuse the temperature recorded at the maximum of the endothermic melting peak (Tmax) with the maximum melting point temperature of the ice in solution, because Tm is less or equal to Tmax [119]. In the kinetic phenomena being influenced by the mass involved in the melting process, the comparison of the Tmax values becomes difficult between the series for which the mass of sample studied is different and in which the mass of water in solution concerned by the melting is different.

The actual mass concentrations of COS in the solutions that were studied are presented in **Table 3**. In the following analyses, only the actual mass concentration of COS is considered for a given compound. However, to simplify the notation in thermograms, the hydrated powdered mass concentration of COS is used to desig-

The amounts of ice formed in solution were estimated using the determination of the crystallization enthalpy (ΔHc) and the melting enthalpy (ΔHm). To estimate these amounts, the peak areas were calculated using a sigmoidal curve baseline (with the Pyris software 11.1.1). During warming the tangents, allowing the calculation of the sigmoidal line, were positioning, respectively, just before the colloidal

In order to compare the transition enthalpies, a normalization of the measured values to a specified temperature of 0°C was made. As the enthalpy value of the transition of water to ice evolves as a function of temperature, the "latent heat of solidification of supercooled water" (Lf) was obtained at the measured phase change temperature from Boutron's [118] data interpolation. His data were calculated [119] from Angell's [120] specific heat capacity (Cp) of supercooled water and

The normalization of the measured values to the expected values at the specified temperature of 0°C considers that the same proportion of solution transits at the transition temperature (Tt) (ΔHmeas(Tt)/Lf(Tt)) and at 0°C (ΔHnormalized(0°C)/

<sup>Δ</sup>Hnormalizedð Þ¼ 0°C <sup>Δ</sup>Hmeasð Þ Tt <sup>∗</sup> Lfð Þ 0°C

where ΔHnormalized(0°C) is the transition enthalpy of the sample normalized to

), ΔHmeas(Tt) is the transition enthalpy of the sample measured at a

During cooling, to analyze the crystallization enthalpies, the nucleation temper-

ature (Tn) was considered as the transition temperature (i.e., Tt = Tn). During warming, to analyze the melting enthalpies, the temperature of the summit of the melting peak (Tmax) was considered as the transition temperature (i.e., Tt = Tmax). These are approximations because for a mixture there is not only one transition

When a non-hydrated inert mass is added in a solution, a lowering of the enthalpies of crystallization and melting is expected since this addition decreases, in proportion, the mass of water present in solution. The term "inert" refers to a product that does not influence the transition enthalpies (i.e., a product that does not transit and does not influence the transition of other products). The theoretical link between the decrease of the transition enthalpies and the decrease of the mass proportion of water in solution is given by Eq. (3). The comparison of a theoretical value with a measured one allows estimating the mass of water which transits in comparison to the mass of water present within the solution. Experimental values

Lfð Þ Tt

), Lf(Tt) is the transition enthalpy of a pure water

), and Lf(0°C) is the transition

) (J.g�<sup>1</sup> ). (2)

from Weast's Hand Book's [121] Cp of ice at different temperatures.

nate the solutions.

*7.1.3 Normalization of the transition enthalpies to 0°C*

*Cryopreservation - Current Advances and Evaluations*

transition and just after the melting peak.

Lf(0°C)). This equality leads to Eq. (2):

sample measured at a transition temperature Tt (J.g�<sup>1</sup>

enthalpy of a pure water sample measured at 0°C (= 333.4 J.g�<sup>1</sup>

*7.1.4 Comparison of measured and calculated transition enthalpies*

*with the addition of an inert mass*

transition temperature Tt (J.g�<sup>1</sup>

0°C (J.g�<sup>1</sup>

temperature.

**14**

Since the Tmax values cannot be compared and can hardly be associated only with a cryoscopic phenomenon, we have not realized this comparison. We used an alternative method for estimating the cryoscopic effect based on the shape of the thermograms. It consists in the comparison of the size and the peaks spread with temperature [119].

the solution does not disturb further the ability of water molecules to crystallize. Thus, the COS presence does not alter either the amount of bounded water or the

*The Use of Chitooligosaccharides in Cryopreservation: Discussion of Concept and First…*

reduced, as indicated by the smaller normalized values of ΔHc and ΔHm. This decrease in crystallization and melting enthalpies implies a decrease in the amount of ice formed. Because normalized values are lower than the calculated, we argue that it is the materialization of the capability of COS to stabilize the remaining solution and to limit the crystallization of a part of the water molecules in the

**7.3 Cryoscopic depression induced by the addition of COS**

For the highest COS concentrations, the amount of water that can crystallize is

COS, therefore, serve the purpose of reducing ice formation during cryopreservation procedures. However, their mode of action and its consequences for cells

As could be seen in **Figure 2**, when COS is added, the maximum of the heat flow related to the melting endothermic peaks appears to be smaller, and the peaks are

When there is little ice in the sample, the effects responsible for the temperature difference between Tm and Tmax decrease. Thus, the decrease in the amount of ice formed when the amount of COS increases (cf. **Figure 1**) could explain the lowering of Tmax (cf. **Figure 2**) without cryoscopic effect. When there is a smaller amount of ice and fewer phenomena that may imply the shift of the temperature of the

*Zoom on the bottom of the melting peaks obtained for the mixture: IMV + 5% (v/v) Me2SO + [COS10\_0]. Observation of the COS influence on the shape of the melting peaks. Protocol, 2. Sample volume = 10 μL.*

*up from* �*100 to* �*80°C and then lined up at 0.0 mW. The mass concentrations of the studied hydrated powder*

*thermograms, a large one which is associated to the melting of the ice previously crystallized within the sample and a small one which is an anomaly. This anomaly of melting is systematically present during the analysis of slow-freezing solutions, linked to the melting of the previously condensed and crystallized ambient humidity of the air encapsulated within the sample pan. This humidity could be from the previous ambient air in the laboratory or from the equilibration at the vapor pressure of water between the encapsulated air and the sample*

*. Data range, normal. All cooling and warming thermograms were straightened*

*. Two endothermic peaks are observable on each of these*

quantity of bulk water that vitrified.

*DOI: http://dx.doi.org/10.5772/intechopen.89162*

remaining solution.

**Figure 2.**

**17**

*Warming rate = +2.5°C.min*�*<sup>1</sup>*

*are equal to 0, 12.5, 25, 100, and 200 mg.mL*�*<sup>1</sup>*

*aqueous solution or from a combination of both.*

deserve to be studied deeper.

shifted toward the low temperatures.

## **7.2 Phase transition analysis of crystallizable water**

The calculated values of the crystallization and melting enthalpies of the solutions were normalized to 0°C and represented in **Figure 1**. The calculation of the phase change enthalpy expected following the addition of an inert mass was performed in the range of the concentrations studied, using Eqs. (3) and (4). These values were plotted (**Figure 1**), with measured enthalpy values (normalized to 0°C), as a function of the initial COS concentration.

A sharp decrease in the ΔHc and ΔHm values is observed as a function of the mass concentration of COS introduced, and this decrease seems affine. For the same mass concentration of non-hydrated powder studied, the differences between ΔHc and ΔHm are small. Until 50 mg.mL�<sup>1</sup> , a good correlation is observable between the calculated mass enthalpies following the addition of a non-hydrated inert mass and the measured mass enthalpies in the presence of COS (cf. **Figure 1**). COS molecules added in the solution can replace part of the water molecules present in the solution and thus reduce the amount of crystallizable water in mass proportion (as materialized by the orange line in **Figure 1**). To play an additional role, the CPA molecules must either bind to a portion of the water molecules to prevent their crystallization or promote the glass-forming tendency of free water during cooling. Based on **Figure 1**, COS do not seem to have significant effects, although these effects increase for the higher COS concentration (> 100 mg.mL�<sup>1</sup> ).

For the smallest COS concentrations, if the COS could bind to a part of the water, or highly favor the vitrification, then a difference between calculated enthalpies and experimental enthalpies or a difference between the crystallization enthalpies and the melting enthalpies (for a nominative mass of COS introduced in solution) would have been expected. These results imply that the addition of COS in

## **Figure 1.**

*Crystallization and melting enthalpies, normalized to 0°C, for the different solutions containing COS. Here the calculated transition enthalpies are also materialized in the case of the addition of a non-hydrated inert mass in solution. The standard deviations are calculated according to the three data obtained for each value. The standard deviation of the calculated enthalpy is the standard deviation of the measured value without COS.*

*The Use of Chitooligosaccharides in Cryopreservation: Discussion of Concept and First… DOI: http://dx.doi.org/10.5772/intechopen.89162*

the solution does not disturb further the ability of water molecules to crystallize. Thus, the COS presence does not alter either the amount of bounded water or the quantity of bulk water that vitrified.

For the highest COS concentrations, the amount of water that can crystallize is reduced, as indicated by the smaller normalized values of ΔHc and ΔHm. This decrease in crystallization and melting enthalpies implies a decrease in the amount of ice formed. Because normalized values are lower than the calculated, we argue that it is the materialization of the capability of COS to stabilize the remaining solution and to limit the crystallization of a part of the water molecules in the remaining solution.

COS, therefore, serve the purpose of reducing ice formation during cryopreservation procedures. However, their mode of action and its consequences for cells deserve to be studied deeper.

## **7.3 Cryoscopic depression induced by the addition of COS**

As could be seen in **Figure 2**, when COS is added, the maximum of the heat flow related to the melting endothermic peaks appears to be smaller, and the peaks are shifted toward the low temperatures.

When there is little ice in the sample, the effects responsible for the temperature difference between Tm and Tmax decrease. Thus, the decrease in the amount of ice formed when the amount of COS increases (cf. **Figure 1**) could explain the lowering of Tmax (cf. **Figure 2**) without cryoscopic effect. When there is a smaller amount of ice and fewer phenomena that may imply the shift of the temperature of the

### **Figure 2.**

Since the Tmax values cannot be compared and can hardly be associated only with a cryoscopic phenomenon, we have not realized this comparison. We used an alternative method for estimating the cryoscopic effect based on the shape of the thermograms. It consists in the comparison of the size and the peaks spread with

The calculated values of the crystallization and melting enthalpies of the solutions were normalized to 0°C and represented in **Figure 1**. The calculation of the phase change enthalpy expected following the addition of an inert mass was performed in the range of the concentrations studied, using Eqs. (3) and (4). These values were plotted (**Figure 1**), with measured enthalpy values (normalized to

A sharp decrease in the ΔHc and ΔHm values is observed as a function of the mass concentration of COS introduced, and this decrease seems affine. For the same mass concentration of non-hydrated powder studied, the differences between ΔHc

calculated mass enthalpies following the addition of a non-hydrated inert mass and the measured mass enthalpies in the presence of COS (cf. **Figure 1**). COS molecules added in the solution can replace part of the water molecules present in the solution and thus reduce the amount of crystallizable water in mass proportion (as materialized by the orange line in **Figure 1**). To play an additional role, the CPA molecules must either bind to a portion of the water molecules to prevent their crystallization or promote the glass-forming tendency of free water during cooling. Based on **Figure 1**, COS do not seem to have significant effects, although these effects

For the smallest COS concentrations, if the COS could bind to a part of the water, or highly favor the vitrification, then a difference between calculated enthalpies and experimental enthalpies or a difference between the crystallization enthalpies and the melting enthalpies (for a nominative mass of COS introduced in solution) would have been expected. These results imply that the addition of COS in

*Crystallization and melting enthalpies, normalized to 0°C, for the different solutions containing COS. Here the calculated transition enthalpies are also materialized in the case of the addition of a non-hydrated inert mass in solution. The standard deviations are calculated according to the three data obtained for each value. The standard deviation of the calculated enthalpy is the standard deviation of the measured value without COS.*

, a good correlation is observable between the

).

**7.2 Phase transition analysis of crystallizable water**

*Cryopreservation - Current Advances and Evaluations*

0°C), as a function of the initial COS concentration.

increase for the higher COS concentration (> 100 mg.mL�<sup>1</sup>

and ΔHm are small. Until 50 mg.mL�<sup>1</sup>

temperature [119].

**Figure 1.**

**16**

*Zoom on the bottom of the melting peaks obtained for the mixture: IMV + 5% (v/v) Me2SO + [COS10\_0]. Observation of the COS influence on the shape of the melting peaks. Protocol, 2. Sample volume = 10 μL. Warming rate = +2.5°C.min*�*<sup>1</sup> . Data range, normal. All cooling and warming thermograms were straightened up from* �*100 to* �*80°C and then lined up at 0.0 mW. The mass concentrations of the studied hydrated powder are equal to 0, 12.5, 25, 100, and 200 mg.mL*�*<sup>1</sup> . Two endothermic peaks are observable on each of these thermograms, a large one which is associated to the melting of the ice previously crystallized within the sample and a small one which is an anomaly. This anomaly of melting is systematically present during the analysis of slow-freezing solutions, linked to the melting of the previously condensed and crystallized ambient humidity of the air encapsulated within the sample pan. This humidity could be from the previous ambient air in the laboratory or from the equilibration at the vapor pressure of water between the encapsulated air and the sample aqueous solution or from a combination of both.*

melting peak summit, the melting peak is smaller, and then, the temperature of the peak's summit is shifted toward low temperatures.

influence of COS on the average size of intercrystalline spans). Further experiments, particularly on their ability to increase the glass-forming tendency of the

*The Use of Chitooligosaccharides in Cryopreservation: Discussion of Concept and First…*

This work was supported by a grant from the Veterinary Campus of Lyon (VetAgro Sup, Marcy l'Etoile, France) and the infrastructure project CRB-Anim

, Magda Teixeira<sup>1</sup>

4 Université de Lyon, IMP 'Ingénierie des Matériaux Polymères', CNRS UMR 5223,

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

, Laurent David<sup>4</sup>

, Amani Moussa<sup>4</sup>

3 Université de Paris, LVTS, Inserm U1148, Paris, France

\*Address all correspondence to: hugodesnos@hotmail.fr

provided the original work is properly cited.

1 Université de Lyon, VetAgro Sup, UPSP ICE 'Interactions Cellules Environnement' and CRB CryAnim (CRB-Anim), Marcy L'Etoile, France

2 Université de Paris, Campus Saint Germain des Près, Paris, France

, Loris Commin<sup>1</sup>

, Samuel Buff<sup>1</sup> and

, Gérard Louis2,3,

remaining solution, should be conducted in the future.

*DOI: http://dx.doi.org/10.5772/intechopen.89162*

**Acknowledgements**

(ANR11-INBS-0003).

**Author details**

Anne Baudot2,3

Stephane Trombotto<sup>4</sup>

Villeurbanne, France

**19**

Hugo Desnos1,2\*, Pierre Bruyère1

However, the offset that is observed at the onset of the peaks cannot be associated with a reduction in the amount of ice that melts during warming. This shift indicates that there is a change in the amount of ice transiting at lower temperatures. The amount of water that melts as a function of temperature is greater at lower temperatures for the solutions with COS (cf. **Figure 2**), even if, in the presence of COS, the total amount of ice previously formed is lower. This phenomenon is the materialization of a slight cryoscopic effect linked to the addition of COS in solution. This effect is, however, difficult to quantify and remains below 0.5°C (cf. **Figure 2**).

The presence of a weak cryoscopic depression associated with the addition of COS in the IMV + 5% (v/v) Me2SO solution involves the addition of COS having little or no influence on the osmolality of the studied solutions. Further verifications will nevertheless have to be made to verify these results, especially by studying the characteristics of the mixture: "H2O-COS." Based on these preliminary findings, we will consider that COS compounds can be added to cryopreservation solutions without fear of a significant increase or acceleration of cell dehydration.

## **8. Conclusion on the COS usage for cryopreservation**

According to this first thermodynamic characterization, the use of COS as CPA is debatable, as are their cryoprotective capabilities.

COS appear to be biocompatible in solution, and no disruption to mouse embryo development has occurred when COS were evaluated. COS have a significant influence on lowering the amount of crystallizable ice in solution, without participating in cell dehydration. Indeed, their addition in solution replaces a significant part of water, without significant osmotic effect. This is an interesting use in slow-freezing cryopreservation since it makes it possible to reduce the mechanical damage associated with the formation of ice, without change in the equilibrium of the osmotic balance with the intracellular media. We can thus assume that the use of such a product does not promote the mechanisms of dehydration of the intracellular medium.

However, COS do not seem able to influence the proportion of IMV + 5% (v/v) Me2SO solution remaining liquid during freezing. As a result, they cannot participate in the main protections associated with CPA. Using these compounds without any other CPA has, therefore, to be discarded. COS do not participate in the equilibration of osmotic pressures between intra- and extracellular media. Thus, they dehydrate the overconcentrated residual solution, without modifying the mass proportion of crystallized water and without dehydrating the cells. They act only in the extracellular medium by inducing a steric hindrance and by increasing the solution viscosity. They seem to contribute to the stabilization of the extracellular noncrystalline medium.

Combined with penetrating cryoprotectants, we assume that COS can promote a decrease in the use of penetrating CPA while ensuring the successful vitrification of intercrystalline spans. Nevertheless, several questions remain about the action of COS in solution. It is particularly possible to question their interactions with water molecules. Similarly, it is difficult to know how these compounds interact with Me2SO. In order to further characterize the role of these compounds in water solution, it will be necessary to characterize them in simpler binary solutions composed of COS and water. It would also be necessary to study the influence of COS on the ice crystal organization in cryopreserved samples (notably to study the

*The Use of Chitooligosaccharides in Cryopreservation: Discussion of Concept and First… DOI: http://dx.doi.org/10.5772/intechopen.89162*

influence of COS on the average size of intercrystalline spans). Further experiments, particularly on their ability to increase the glass-forming tendency of the remaining solution, should be conducted in the future.

## **Acknowledgements**

melting peak summit, the melting peak is smaller, and then, the temperature of the

However, the offset that is observed at the onset of the peaks cannot be associated with a reduction in the amount of ice that melts during warming. This shift indicates that there is a change in the amount of ice transiting at lower temperatures. The amount of water that melts as a function of temperature is greater at lower temperatures for the solutions with COS (cf. **Figure 2**), even if, in the presence of COS, the total amount of ice previously formed is lower. This phenomenon is the materialization of a slight cryoscopic effect linked to the addition of COS in solution. This effect is, however, difficult to quantify and remains below 0.5°C

The presence of a weak cryoscopic depression associated with the addition of COS in the IMV + 5% (v/v) Me2SO solution involves the addition of COS having little or no influence on the osmolality of the studied solutions. Further verifications will nevertheless have to be made to verify these results, especially by studying the characteristics of the mixture: "H2O-COS." Based on these preliminary findings, we will consider that COS compounds can be added to cryopreservation solutions without fear of a significant increase or acceleration of cell dehydration.

According to this first thermodynamic characterization, the use of COS as CPA

COS appear to be biocompatible in solution, and no disruption to mouse embryo development has occurred when COS were evaluated. COS have a significant influence on lowering the amount of crystallizable ice in solution, without participating in cell dehydration. Indeed, their addition in solution replaces a significant part of water, without significant osmotic effect. This is an interesting use in slow-freezing cryopreservation since it makes it possible to reduce the mechanical damage associated with the formation of ice, without change in the equilibrium of the osmotic balance with the intracellular media. We can thus assume that the use of such a product does not promote the mechanisms of dehydration of the intracellular

However, COS do not seem able to influence the proportion of IMV + 5% (v/v) Me2SO solution remaining liquid during freezing. As a result, they cannot participate in the main protections associated with CPA. Using these compounds without any other CPA has, therefore, to be discarded. COS do not participate in the equilibration of osmotic pressures between intra- and extracellular media. Thus, they dehydrate the overconcentrated residual solution, without modifying the mass proportion of crystallized water and without dehydrating the cells. They act only in the extracellular medium by inducing a steric hindrance and by increasing the solution viscosity. They seem to contribute to the stabilization of the extracellular

Combined with penetrating cryoprotectants, we assume that COS can promote a decrease in the use of penetrating CPA while ensuring the successful vitrification of intercrystalline spans. Nevertheless, several questions remain about the action of COS in solution. It is particularly possible to question their interactions with water molecules. Similarly, it is difficult to know how these compounds interact with Me2SO. In order to further characterize the role of these compounds in water solution, it will be necessary to characterize them in simpler binary solutions composed of COS and water. It would also be necessary to study the influence of COS on

the ice crystal organization in cryopreserved samples (notably to study the

**8. Conclusion on the COS usage for cryopreservation**

is debatable, as are their cryoprotective capabilities.

peak's summit is shifted toward low temperatures.

*Cryopreservation - Current Advances and Evaluations*

(cf. **Figure 2**).

medium.

**18**

noncrystalline medium.

This work was supported by a grant from the Veterinary Campus of Lyon (VetAgro Sup, Marcy l'Etoile, France) and the infrastructure project CRB-Anim (ANR11-INBS-0003).

## **Author details**

Hugo Desnos1,2\*, Pierre Bruyère1 , Magda Teixeira<sup>1</sup> , Loris Commin<sup>1</sup> , Gérard Louis2,3, Stephane Trombotto<sup>4</sup> , Amani Moussa<sup>4</sup> , Laurent David<sup>4</sup> , Samuel Buff<sup>1</sup> and Anne Baudot2,3

1 Université de Lyon, VetAgro Sup, UPSP ICE 'Interactions Cellules Environnement' and CRB CryAnim (CRB-Anim), Marcy L'Etoile, France

2 Université de Paris, Campus Saint Germain des Près, Paris, France

3 Université de Paris, LVTS, Inserm U1148, Paris, France

4 Université de Lyon, IMP 'Ingénierie des Matériaux Polymères', CNRS UMR 5223, Villeurbanne, France

\*Address all correspondence to: hugodesnos@hotmail.fr

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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cryopreservation. Human Reproduction [Internet]. 1999;**14**(4):1013-1021. Available from: http://www.ncbi.nlm.

ncomms4244

[87] Fonseca F, Meneghel J, Cenard S, Passot S, Morris GJ. Determination of Intracellular vitrification temperatures for unicellular micro organisms under

cryopreservation. PLoS One [Internet]. 2016;**11**(4):1-19. 10.1371/journal.

[88] Browne RK, Clulow J, Mahony M. The effect of saccharides on the postthaw recovery of cane toad (*Bufo marinus*) spermatozoa. Cryo Letters [Internet]. 2002;**23**(2):121-128. Available from: http://www.ncbi.nlm.

[89] Huang H, Choi JK, Rao W, Zhao S, Agarwal P, Zhao G, et al. Alginate hydrogel microencapsulation inhibits devitrification and enables large-volume low-CPA cell vitrification. Advanced Functional Materials. 2015;**25**(44):

[90] Meneghel J, Passot S, Cenard S, Réfrégiers M, Jamme F, Fonseca F. Subcellular membrane fluidity of Lactobacillus delbrueckii subsp.

10.1007/s00253-017-8444-9

rsif.2014.0069

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bulgaricus under cold and osmotic stress. Applied Microbiology and Biotechnology [Internet]. 2017;**101**(18):6907-6917. DOI:

[91] Kent B, Hunt T, Darwish TA, Hauß T, Garvey CJ, Bryant G. Localization of trehalose in partially hydrated DOPC bilayers: Insights into cryoprotective mechanisms. Journal of The Royal Society Interface [Internet]. 2014;**11** (95):20140069. DOI: 10.1098/

[92] Deller RC, Vatish M, Mitchell DA, Gibson MI. Synthetic polymers enable non-vitreous cellular cryopreservation by reducing ice crystal growth during thawing. Nature Communications

[101] Seki S, Mazur P. Effect of warming rate on the survival of vitrified mouse oocytes and on the recrystallization of intracellular Ice1. Biology of Reproduction [Internet]. 2008;**79**(4):727-737. DOI: 10.1095/biolreprod.108.069401

[102] Generalic E. Acetyl glucosamine [Internet]. Glossary, Croatian-English Chemistry Dictionary. 2017. Available from: https://glossary.periodni.com/dic tionary.php?en=aceyl+glucosamine

[103] Xia W, Liu P, Zhang J, Chen J. Biological activities of chitosan and chitooligosaccharides. Food Hydrocolloids [Internet]. 2011;**25**(2): 170-179. DOI: 10.1016/j. foodhyd.2010.03.003

[104] Kim SK, Rajapakse N. Enzymatic production and biological activities of chitosan oligosaccharides (COS): A review. Carbohydrate Polymers. 2005; **62**(4):357-368

[105] Aam BB, Heggset EB, Norberg AL, Sørlie M, Vårum KM, Eijsink VGH. Production of chitooligosaccharides and their potential applications in medicine. Marine Drugs. 2010;**8**(5):1482-1517

[106] Escobedo-Lozano AY, Domard A, Velázquez CA, Goycoolea FM, Argüelles-Monal WM. Physical properties and antibacterial activity of chitosan/acemannan mixed systems. Carbohydrate Polymers [Internet]. 2015;**115**:707-714. DOI: 10.1016/j. carbpol.2014.07.064

[107] Peter MG. Applications and environmental aspects of chitin and chitosan. Journal of Macromolecular Science, Part A [Internet]. 1995;**32**(4): 629-640. DOI: 10.1080/ 10601329508010276

[108] Adekunle KF. Bio-based polymers for technical applications: A review— Part 2. Open Journal of Polymer Chemistry [Internet]. 2014;**04**(04):95- 101. DOI: 10.4236/ojpchem.2014.44011

[109] Teixeira M, Buff S, Desnos H, Loiseau C, Bruyère P, Joly T, et al. Ice nucleating agents allow embryo freezing without manual seeding. Theriogenology [Internet]. 2017; **104**(104):173-178 Available from: http:// linkinghub.elsevier.com/retrieve/pii/ S0093691X17303977

[110] Moussa A, Crépet A, Ladavière C, Trombotto S. Reducing-end "clickable" functionalizations of chitosan oligomers for the synthesis of chitosan-based diblock copolymers. Carbohydrate Polymers [Internet]. 2019;**219**:387-394. Available from: https://linkinghub.else vier.com/retrieve/pii/ S0144861719304783

[111] Becerra J, Sudre G, Royaud I, Montserret R, Verrier B, Rochas C, et al. Tuning the hydrophilic/hydrophobic balance to control the structure of chitosan films and their protein release behavior. AAPS PharmSciTech [Internet]. 2017;**18**(4):1070-1083. DOI: 10.1208/s12249-016-0678-9

[112] Dhawade PP, Jagtap RN. Characterization of the glass transition temperature of chitosan and its oligomers by temperature modulated differential scanning calorimetry. Advances in Applied Science Research [Internet]. 2012;**3**(3):1372-1382. Available from: www.pelagiaresearchlib rary.com

[113] Payet L. Viscoelasticite et structure de gels à base de chitosane - relations avec les propriétés diffusionnelles de macromolécules dans ces biogels. Solutions. Université Paris 7 - Denis Diderot; 2005

[114] Barros SC, da Silva AA, Costa DB, Costa CM, Lanceros-Méndez S, Maciavello MNT, et al. Thermal– mechanical behaviour of chitosan– cellulose derivative thermoreversible hydrogel films. Cellulose. 2015;**22**(3): 1911-1929

[115] Julkapli NM, Akil HM. Influence of a plasticizer on the mechanical properties of kenaf-filled chitosan bio-composites. Polymer-Plastics Technology and Engineering. 2010;**49**(9):944-951

[116] El-Hefian EA, Elgannoudi ES, Mainal A, Yahaya AH. Characterization of chitosan in acetic acid: Rheological and thermal studies. Turkish Journal of Chemistry. 2010;**34**(1):47-56

[117] Desnos H, Baudot A, Teixeira M, Louis G, Commin L, Buff S, et al. Ice induction in DSC experiments with Snomax®. Thermochimica Acta [Internet]. 2018;**667**:193-206 Available from: https://linkinghub.elsevier.com/re trieve/pii/S0040603118305938

[118] Boutron P. More accurate determination of the quantity of ice crystallized at low cooling rates in the glycerol and 1,2-propanediol aqueous solutions: Comparison with equilibrium. Cryobiology. 1984;**21**(2):183-191

[119] Desnos H. Amélioration des procédures de cryoconservation de type congélation-lente par simulation et caractérisation des effets de composés chitooligosaccharides. University Lyon 1; 2019

[120] Angell CA, Shuppert J, Tucker JC. Anomalous properties of supercooled water. Heat capacity, expansivity, and proton magnetic resonance chemical shift from 0 to �38°C. The Journal of Physical Chemistry [Internet]. 1973; **77**(26):3092-3099. DOI: 10.1021/ j100644a014

[121] Weast RC. CRC Handbook of Chemistry and Physics. 60th ed. In: Weast RC, editor. CRC Press; 1979

**29**

**Chapter 2**

**Abstract**

aggregation

**1. Introduction**

Cryoprotection of Platelets by

*Mark D. Scott, Nobu Nakane and Elisabeth Maurer-Spurej*

Unlike red blood cells (RBC) which are stored at 4°C, platelets are stored at 22–24°C (room temperature) due to biophysical and biochemical changes induced by cold temperatures aggregately known as the 'cold storage lesion' (CSL). However, 22°C storage greatly increases the risk of microbial growth, thus limiting the safe storage of platelets to only 5–7 days (versus 42 days for RBC). Consequent to the short shelf life of platelets, blood services face chronic shortages of these life-saving cells. To overcome both the risk of microbial contamination and the constrained supplies of platelets, renewed research into attenuating the CSL and/or determining where cold stored platelets are clinically suitable are ongoing. In this chapter, we show that the covalent grafting of methoxypolyethylene glycol (mPEG), a biocompatible polymer, to the membrane of platelets attenuates the CSL. Moreover, the grafted mPEG serves as a potent cryoprotectant allowing platelets to be stored at 4°C, or frozen at −20°C, while retaining normal platelet counts and biologic function. The successful development of platelet PEGylation may provide a means by which the cold storage of platelets can be achieved with a minimal loss of platelet

quality while improving both platelet microbial safety and inventory.

and initiate coagulation to stop bleeding (*i.e.* haemostasis).

**Keywords:** cryopreservation, cryoprotection, platelets, blood banking, cold storage, PEGylation, immunocamouflage, methoxypoly(ethylene glycol), polymer,

Platelet adhesion and aggregation at the site of vascular injury are key events required for normal vascular homeostasis and wound repair. [1–4] Platelets are produced from megakaryocytes in the bone marrow and, while lacking a nucleus, contain a number of specialized granules such as alpha-granules and dense granules. Normal, resting platelets have a discoid morphology which changes upon activation to 'spiny spheres' arising from the formation of pseudopodia. This shape change coincides with the rearrangement of the actin cytoskeleton. Upon activation, platelets adhere to the subendothelium at sites of vascular injury, aggregate

Consequent to this essential role, platelet transfusions have evolved as a crucial therapeutic tool in the treatment of a large number of diverse clinical conditions including acute bleeding, surgery, treatment of a variety of cancers, patients with platelet abnormalities and autoimmune diseases such as Idiopathic Thrombocytopenic Purpura (ITP) [5]. To meet the increasing clinical needs, blood systems within developed countries produce in excess of 5,000,000 transfusion

Grafted Polymers

## **Chapter 2**

[114] Barros SC, da Silva AA, Costa DB,

*Cryopreservation - Current Advances and Evaluations*

[121] Weast RC. CRC Handbook of Chemistry and Physics. 60th ed. In: Weast RC, editor. CRC Press; 1979

[115] Julkapli NM, Akil HM. Influence of a plasticizer on the mechanical properties of kenaf-filled chitosan bio-composites. Polymer-Plastics Technology and Engineering. 2010;**49**(9):944-951

[116] El-Hefian EA, Elgannoudi ES, Mainal A, Yahaya AH. Characterization of chitosan in acetic acid: Rheological and thermal studies. Turkish Journal of

[117] Desnos H, Baudot A, Teixeira M, Louis G, Commin L, Buff S, et al. Ice induction in DSC experiments with Snomax®. Thermochimica Acta [Internet]. 2018;**667**:193-206 Available from: https://linkinghub.elsevier.com/re

Chemistry. 2010;**34**(1):47-56

trieve/pii/S0040603118305938

[118] Boutron P. More accurate determination of the quantity of ice crystallized at low cooling rates in the glycerol and 1,2-propanediol aqueous solutions: Comparison with equilibrium.

Cryobiology. 1984;**21**(2):183-191

[119] Desnos H. Amélioration des procédures de cryoconservation de type congélation-lente par simulation et caractérisation des effets de composés chitooligosaccharides. University Lyon

[120] Angell CA, Shuppert J, Tucker JC. Anomalous properties of supercooled water. Heat capacity, expansivity, and proton magnetic resonance chemical shift from 0 to �38°C. The Journal of Physical Chemistry [Internet]. 1973; **77**(26):3092-3099. DOI: 10.1021/

1; 2019

j100644a014

**28**

Costa CM, Lanceros-Méndez S, Maciavello MNT, et al. Thermal– mechanical behaviour of chitosan– cellulose derivative thermoreversible hydrogel films. Cellulose. 2015;**22**(3):

1911-1929

## Cryoprotection of Platelets by Grafted Polymers

*Mark D. Scott, Nobu Nakane and Elisabeth Maurer-Spurej*

## **Abstract**

Unlike red blood cells (RBC) which are stored at 4°C, platelets are stored at 22–24°C (room temperature) due to biophysical and biochemical changes induced by cold temperatures aggregately known as the 'cold storage lesion' (CSL). However, 22°C storage greatly increases the risk of microbial growth, thus limiting the safe storage of platelets to only 5–7 days (versus 42 days for RBC). Consequent to the short shelf life of platelets, blood services face chronic shortages of these life-saving cells. To overcome both the risk of microbial contamination and the constrained supplies of platelets, renewed research into attenuating the CSL and/or determining where cold stored platelets are clinically suitable are ongoing. In this chapter, we show that the covalent grafting of methoxypolyethylene glycol (mPEG), a biocompatible polymer, to the membrane of platelets attenuates the CSL. Moreover, the grafted mPEG serves as a potent cryoprotectant allowing platelets to be stored at 4°C, or frozen at −20°C, while retaining normal platelet counts and biologic function. The successful development of platelet PEGylation may provide a means by which the cold storage of platelets can be achieved with a minimal loss of platelet quality while improving both platelet microbial safety and inventory.

**Keywords:** cryopreservation, cryoprotection, platelets, blood banking, cold storage, PEGylation, immunocamouflage, methoxypoly(ethylene glycol), polymer, aggregation

## **1. Introduction**

Platelet adhesion and aggregation at the site of vascular injury are key events required for normal vascular homeostasis and wound repair. [1–4] Platelets are produced from megakaryocytes in the bone marrow and, while lacking a nucleus, contain a number of specialized granules such as alpha-granules and dense granules. Normal, resting platelets have a discoid morphology which changes upon activation to 'spiny spheres' arising from the formation of pseudopodia. This shape change coincides with the rearrangement of the actin cytoskeleton. Upon activation, platelets adhere to the subendothelium at sites of vascular injury, aggregate and initiate coagulation to stop bleeding (*i.e.* haemostasis).

Consequent to this essential role, platelet transfusions have evolved as a crucial therapeutic tool in the treatment of a large number of diverse clinical conditions including acute bleeding, surgery, treatment of a variety of cancers, patients with platelet abnormalities and autoimmune diseases such as Idiopathic Thrombocytopenic Purpura (ITP) [5]. To meet the increasing clinical needs, blood systems within developed countries produce in excess of 5,000,000 transfusion

doses annually [6]. However, demand for platelets continues to increase annually while the rate of blood/platelet donations are actually declining leading to an inventory that is chronically constrained [7].

The constraint of platelet inventory is in large part due to an inability to safely store platelet products for greater than 5–7 days. Historically, platelets, like red blood cells (RBC), were stored at 4°C and successfully used clinically. However, multiple studies from the late 1960s to the early 1970s demonstrated that 4°C (*i.e.* cold) storage of platelets resulted in significantly reduced *in vivo* survival times compared to platelets stored at 22°C (warm storage) or endogenously produced platelets (2–4 vs. 7–9 vs. 10–12 days, respectively) [8–17]. The observed loss of *in vivo* viability and *in vitro* morphology and function was termed the platelet cold storage lesion (CSL) and resulted in the change in standard blood banking practice to storing platelets at 22–24°C by the early 1970s.

The CSL is multi-dimensional and is best characterized as the sum of all the deleterious changes in platelet morphology, biochemistry and function that arise from the time the blood is withdrawn to the time the cold-stored platelets are transfused. The CSL is characterized, in part, by loss of discoid shape (*i.e.* abnormal morphological forms), decreased mean platelet volume, increased size

## **Figure 1.**

*Schematic view of the prevention of the platelet 'cold storage lesion' (CSL) by membrane PEGylation. Panel A: The normal discoid shape of platelets is lost as platelets are cooled below ~18°C. As a consequence of cooling, pseudopodia formation occurs leading to microaggregation of platelets (photo insert). Additionally, membrane proteins such as GP1b-IX aggregate on the surface. These changes lead to both mechanical and immunological clearance from the circulation. Panel B: PEGylation of platelets reduces both the shape change (*e.g. *fewer pseudopodia) and prevents microaggregation of the cold stored platelets. Consequent to the attenuation of the CSL lesions by the grafted mPEG, platelets can be stored for extended periods (> 7 days) at 4 or −80°C thereby improving platelet inventory and supply management while reducing the platelet discard rates. As noted, cold storage also significantly reduces the risk of microbial growth thus, potentially, improving transfusion safety.*

**31**

*Cryoprotection of Platelets by Grafted Polymers DOI: http://dx.doi.org/10.5772/intechopen.89272*

tion times [8–16].

demand for platelet products.

**2. What was 'OLD' is 'NEW' again**

prolonged cold storage (*e.g.* >48 hours) [27].

heterogeneity, pseudopodia formation, increased release of platelet α-granules and cytosolic proteins, altered surface protein expression (*e.g.*, glycoproteins such as GP1b-IX), increased procoagulant activity, aggregate formation, and reduced platelet counts—all of which are also characteristic of platelet activation (**Figure 1A**) [8–16, 18, 19]. In contrast, warm storage of platelets maintained platelet morphology, activation potential and greatly improved *in vivo* circula-

However, the warm storage of platelets was not without risk as it was demonstrated that warm storage significantly increased the risk for bacterial growth should bacteria be introduced to the platelet unit during collection [20–24]. Indeed, numerous North American screening studies have indicated that approximately 1/3500 platelet units (primarily platelet rich plasma; PRP) are bacterially contaminated posing a potential hazard to already at-risk patients [23, 25, 26]. Consequent to this risk, multiple blood systems have implemented costly universal bacteriologic screening of donor platelets. Hence, development of new technologies to improve both platelet safety and inventories will be crucial in meeting the ever increasing

Consequent to the clinical demand and supply chain issues, several studies over the last several years have re-explored the potential use of 'cold-stored' platelets. Initial excitement regarding cold-stored platelets arose in 2003, Hoffmeister et al. investigated the mechanism(s) underlying the CSL and experimentally demonstrated that the shape change alone induced by cold storage itself did not result in poor platelet survival in a murine model [18, 19]. Instead, Hoffmeister et al. hypothesized that poor platelet survival resulted from an irreversible membrane clustering of alpha subunits of glycoprotein Ib (GPIbα). Their studies reported that exposed, terminal, beta-linked N-acetylglucosamine (βGlcNAc) residues on clustered GPIbα were recognized by the lectin domain of type 3 complement receptors (CR3; αMβ2; CD11b/CD18) on liver and splenic macrophages. This immunorecognition resulted in the rapid clearance of cold stored donor platelets via phagocytosis. Hoffmeister also demonstrate that phagocytosis of briefly chilled murine platelets could be inhibited and *in vivo* survival prolonged by enzymatically galactosylating the terminal βGlcNAc residues on GPIbα. These findings led them to propose that enzymatically masking the exposed ßGlcNAc residues on the N-glycans of the clustered GPIbα molecules by galactosylation would allow for the cold storage of human platelets without adversely affecting platelet function. However, enthusiasm for glycosylated platelets subsided when subsequent studies by Wandall et al. demonstrated that galactosylation alone did NOT protect murine or human platelets from

More recently, the 'old' (1960) has become 'new' (2019) as transfusion scientists have begun to reexamine the clinical utility of platelets stored at 4°C. Indeed, the original 1960's/70's studies that initially discovered the platelet CSL, also reported that 'cold-stored' platelets were still effective *in vivo* in preventing acute blood loss. Hence, current clinical studies are investigating the use of 4°C stored platelets for at least some transfusion demands [28–45]. In general, recent studies suggest that, while these 'old technology' cold-stored platelets could be of benefit for acute haemostatic transfusion needs, cold stored platelets still exhibit morphological changes and poor *in vivo* survival making them unlikely candidates for chronic replacement therapy in patients with already accelerated platelet clearance or as therapeutics for

patients with cancer or who have undergone bone marrow transplantation.

## *Cryoprotection of Platelets by Grafted Polymers DOI: http://dx.doi.org/10.5772/intechopen.89272*

*Cryopreservation - Current Advances and Evaluations*

to storing platelets at 22–24°C by the early 1970s.

tory that is chronically constrained [7].

doses annually [6]. However, demand for platelets continues to increase annually while the rate of blood/platelet donations are actually declining leading to an inven-

The constraint of platelet inventory is in large part due to an inability to safely store platelet products for greater than 5–7 days. Historically, platelets, like red blood cells (RBC), were stored at 4°C and successfully used clinically. However, multiple studies from the late 1960s to the early 1970s demonstrated that 4°C (*i.e.* cold) storage of platelets resulted in significantly reduced *in vivo* survival times compared to platelets stored at 22°C (warm storage) or endogenously produced platelets (2–4 vs. 7–9 vs. 10–12 days, respectively) [8–17]. The observed loss of *in vivo* viability and *in vitro* morphology and function was termed the platelet cold storage lesion (CSL) and resulted in the change in standard blood banking practice

The CSL is multi-dimensional and is best characterized as the sum of all the deleterious changes in platelet morphology, biochemistry and function that arise from the time the blood is withdrawn to the time the cold-stored platelets are transfused. The CSL is characterized, in part, by loss of discoid shape (*i.e.* abnormal morphological forms), decreased mean platelet volume, increased size

**30**

**Figure 1.**

*Schematic view of the prevention of the platelet 'cold storage lesion' (CSL) by membrane PEGylation. Panel A: The normal discoid shape of platelets is lost as platelets are cooled below ~18°C. As a consequence of cooling, pseudopodia formation occurs leading to microaggregation of platelets (photo insert). Additionally, membrane proteins such as GP1b-IX aggregate on the surface. These changes lead to both mechanical and immunological clearance from the circulation. Panel B: PEGylation of platelets reduces both the shape change (*e.g. *fewer pseudopodia) and prevents microaggregation of the cold stored platelets. Consequent to the attenuation of the CSL lesions by the grafted mPEG, platelets can be stored for extended periods (> 7 days) at 4 or −80°C thereby improving platelet inventory and supply management while reducing the platelet discard rates. As noted, cold storage also significantly reduces the risk of microbial growth thus, potentially, improving transfusion safety.*

heterogeneity, pseudopodia formation, increased release of platelet α-granules and cytosolic proteins, altered surface protein expression (*e.g.*, glycoproteins such as GP1b-IX), increased procoagulant activity, aggregate formation, and reduced platelet counts—all of which are also characteristic of platelet activation (**Figure 1A**) [8–16, 18, 19]. In contrast, warm storage of platelets maintained platelet morphology, activation potential and greatly improved *in vivo* circulation times [8–16].

However, the warm storage of platelets was not without risk as it was demonstrated that warm storage significantly increased the risk for bacterial growth should bacteria be introduced to the platelet unit during collection [20–24]. Indeed, numerous North American screening studies have indicated that approximately 1/3500 platelet units (primarily platelet rich plasma; PRP) are bacterially contaminated posing a potential hazard to already at-risk patients [23, 25, 26]. Consequent to this risk, multiple blood systems have implemented costly universal bacteriologic screening of donor platelets. Hence, development of new technologies to improve both platelet safety and inventories will be crucial in meeting the ever increasing demand for platelet products.

## **2. What was 'OLD' is 'NEW' again**

Consequent to the clinical demand and supply chain issues, several studies over the last several years have re-explored the potential use of 'cold-stored' platelets. Initial excitement regarding cold-stored platelets arose in 2003, Hoffmeister et al. investigated the mechanism(s) underlying the CSL and experimentally demonstrated that the shape change alone induced by cold storage itself did not result in poor platelet survival in a murine model [18, 19]. Instead, Hoffmeister et al. hypothesized that poor platelet survival resulted from an irreversible membrane clustering of alpha subunits of glycoprotein Ib (GPIbα). Their studies reported that exposed, terminal, beta-linked N-acetylglucosamine (βGlcNAc) residues on clustered GPIbα were recognized by the lectin domain of type 3 complement receptors (CR3; αMβ2; CD11b/CD18) on liver and splenic macrophages. This immunorecognition resulted in the rapid clearance of cold stored donor platelets via phagocytosis. Hoffmeister also demonstrate that phagocytosis of briefly chilled murine platelets could be inhibited and *in vivo* survival prolonged by enzymatically galactosylating the terminal βGlcNAc residues on GPIbα. These findings led them to propose that enzymatically masking the exposed ßGlcNAc residues on the N-glycans of the clustered GPIbα molecules by galactosylation would allow for the cold storage of human platelets without adversely affecting platelet function. However, enthusiasm for glycosylated platelets subsided when subsequent studies by Wandall et al. demonstrated that galactosylation alone did NOT protect murine or human platelets from prolonged cold storage (*e.g.* >48 hours) [27].

More recently, the 'old' (1960) has become 'new' (2019) as transfusion scientists have begun to reexamine the clinical utility of platelets stored at 4°C. Indeed, the original 1960's/70's studies that initially discovered the platelet CSL, also reported that 'cold-stored' platelets were still effective *in vivo* in preventing acute blood loss. Hence, current clinical studies are investigating the use of 4°C stored platelets for at least some transfusion demands [28–45]. In general, recent studies suggest that, while these 'old technology' cold-stored platelets could be of benefit for acute haemostatic transfusion needs, cold stored platelets still exhibit morphological changes and poor *in vivo* survival making them unlikely candidates for chronic replacement therapy in patients with already accelerated platelet clearance or as therapeutics for patients with cancer or who have undergone bone marrow transplantation.

## **3. Hypothesis: attenuating the CSL via membrane-grafted mPEG**

Consequent to our earlier work on polymer grafting to intact cells (*e.g.* RBC, lymphocytes), we hypothesized that the polymer induced immunocamouflage of platelet membranes with methoxypoly(ethylene glycol) [mPEG] could prevent or circumvent the immune recognition of cold stored platelets [46–49]. This hypothesis was supported by our previous studies on RBC and leukocytes (White blood cells; WBC) that demonstrated that the grafted polymer prevented cell:cell interactions (*e.g.* RBC Rouleaux formation; Phagocytosis of opsonized RBC; and Lymphocyte:APC) and membrane protein clustering (RBC CR1 aggregation) of the mPEG-modified cells while maintaining normal cellular function [50–71]. Hence, it was hypothesized that mPEG-grafting to platelets would prevent platelet aggregation and the clustering of GPIbα, and phagocytic recognition of the transfused mPEG-platelets (**Figure 1B**). Moreover, because soluble mPEG is a known cryoprotectant, we hypothesized that the grafted polymer might also attenuate other 'cold-induced' mechanical lesions of the CSL induced by cold temperatures and even freezing of donor platelets [72–79]. Indeed, the ability to freeze and recover donor platelets would both greatly increase platelet inventory and potentially expand the use of platelet therapy to geographic locations where platelet therapy is not commonly practiced.

## **4. Polymer engineering of platelets**

All human experiments were done in accordance with the approval of the University of British Columbia Clinical Research Ethics Board and the Canadian Blood Services Ethics Review Board in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki). Following informed consent, fresh platelet rich plasma (PRP) and, in some cases, buffy coat platelets (the standard of care in Canada and Western Europe) were obtained from volunteer donors or the Canadian Blood Services Network Centre for Applied Development (NetCAD) Laboratory (Vancouver, BC). PRP samples are similar to the platelet preparations used clinically and have an advantage of a lower level of manipulation (*e.g.* centrifugation) than buffy coat or apheresis platelets. Care was taken to assure adequate representation of males and females and no individuals were excluded on the basis of age (within acceptable age range of donation of 17–71) or race.

Based on our previous studies, platelets were PEGylated using a semi-automated PEGylation device to maintain a constant platelet:polymer ratio **(Figure 2)** in a micromixing chamber to assure uniform polymer grafting [50, 52, 56, 71]. Platelets were modified with monofunctional (*i.e.* one binding site per chain) mPEG-succinimidyl propionate (SPA-mPEG). Previous studies (not shown) within our laboratories determined that the optimal molecular weights for the PEGylation of platelets were 2–5 kDa which were used in these studies. For comparison of the effects of different linker chemistry, some studies simultaneously examined mPEG-benzotriazolyl carbonate (BTC-mPEG). Both SPA- and BTC-mPEG (Laysan Bio, Inc., Arab, AL, USA) react with protein lysine residues and covalently attach via formation of a stable amide bond (**Figure 2C**). To demonstrate the efficacy of the polymermediated grafting, the mPEG-mediated immunocamouflage of platelet CD9 was assessed by flow cytometry. CD9 is a constitutive tetraspan membrane glycoprotein present on resting platelets that modulates cell adhesion and migration.

Control and PEGylated mini-units (approximately 50 ml/unit; ~500 × 109 /L) of platelets were stored at 4 or 22°C with agitation per Canadian Blood Services standard operating procedures. Storage at −80°C was done separately in the sample

**33**

*Cryoprotection of Platelets by Grafted Polymers DOI: http://dx.doi.org/10.5772/intechopen.89272*

mini-unit blood banking bags. Storage was done for up to 12 days under the prescribed conditions (note: normal storage at 22°C is only allowed for 7 days). Storage bags were sampled aseptically in biosafety cabinets; washing and lysis procedures were performed as described previously [80–82]. Platelet counts were determined

*Production of mPEG-platelets and the SVAmPEG reaction scheme and grafting efficacy. Panel A: semiautomated PEGylation device allowing for control of the mPEG:platelet ratio for uniform grafting levels [71]. Panels B and C: the structure (B) and reaction scheme (C) for activated SVA-mPEG. The SVA-linker chemistry forms a stable amide bond with platelet membrane proteins. Panel D: Fluorescein-conjugated SVAmPEG demonstrated that platelets were uniformly modified using our semi-automated methodology.*

The covalent grafting of mPEG to PRP platelets resulted in the efficient immunocamouflage of CD9 (**Figure 3A**). As demonstrated in **Figure 3A**, virtually 100%

dose effect on the immunocamouflage of CD9. More importantly, the grafted polymer significantly decreased the aggregation of human platelets at 4°C. As microscopically demonstrated in **Figure 3B**, temperature exerted a significant effect on the morphology and microaggregation of control PRP preparations. As anticipated, minimal differences were observed in the control platelets at 37° (*i.e. in vivo* conditions) versus 22°C (normal *in vitro* storage temperature). Importantly, PEGylation with either SCmPEG5000 or BTCmPEG5000 yielded platelets with comparable morphology to the control cells at 37 and 22°C. However, upon thermal transition from 22 to 4°C, control platelets were observed to form significant microaggregates characteristic of the CSL. In stark contrast, neither the SCmPEG5000 nor BTCmPEG5000 modified platelets exhibited any significant microaggregation consequent to the mPEG-mediated inhibition of cell:cell interaction [66, 68]. Morphological analysis of the SCmPEG5000 and BTCmPEG5000 modified platelets suggested that SCmPEG5000 better prevented cold induced shape change relative to the BTC polymer resulting in the SC-linker chemistry

The mPEG-mediated inhibition of cold-induced platelet aggregation was also not a short term effect. As demonstrated in **Figure 4**, unmodified control platelets demonstrated significant shape change, microaggregation, and a dramatic (~30%) decrease in platelet count. In contrast, minimal microaggregation was noted in the PEGylated samples following 12 days storage at 4°C. PEGylated platelets also retained a more discoid shape (though some pseudopod formation was noted). Due to the inhibition of microaggregation and inhibition of activation induced shape change, the mPEG-grafted platelets also resulted in a significantly improved platelet count.

, while the mPEG grafting to the platelets exhibited

using an Advia 120 Hematology Analyzer (Bayer Inc., Toronto, Canada).

**5. Effect of 4°C storage on mPEG-platelets**

of control platelets were CD9<sup>+</sup>

**Figure 2.**

being further explored.

*Cryoprotection of Platelets by Grafted Polymers DOI: http://dx.doi.org/10.5772/intechopen.89272*

**Figure 2.**

*Cryopreservation - Current Advances and Evaluations*

not commonly practiced.

**4. Polymer engineering of platelets**

**3. Hypothesis: attenuating the CSL via membrane-grafted mPEG**

Consequent to our earlier work on polymer grafting to intact cells (*e.g.* RBC, lymphocytes), we hypothesized that the polymer induced immunocamouflage of platelet membranes with methoxypoly(ethylene glycol) [mPEG] could prevent or circumvent the immune recognition of cold stored platelets [46–49]. This hypothesis was supported by our previous studies on RBC and leukocytes (White blood cells; WBC) that demonstrated that the grafted polymer prevented cell:cell interactions (*e.g.* RBC Rouleaux formation; Phagocytosis of opsonized RBC; and Lymphocyte:APC) and membrane protein clustering (RBC CR1 aggregation) of the mPEG-modified cells while maintaining normal cellular function [50–71]. Hence, it was hypothesized that mPEG-grafting to platelets would prevent platelet aggregation and the clustering of GPIbα, and phagocytic recognition of the transfused mPEG-platelets (**Figure 1B**). Moreover, because soluble mPEG is a known cryoprotectant, we hypothesized that the grafted polymer might also attenuate other 'cold-induced' mechanical lesions of the CSL induced by cold temperatures and even freezing of donor platelets [72–79]. Indeed, the ability to freeze and recover donor platelets would both greatly increase platelet inventory and potentially expand the use of platelet therapy to geographic locations where platelet therapy is

All human experiments were done in accordance with the approval of the University of British Columbia Clinical Research Ethics Board and the Canadian Blood Services Ethics Review Board in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki). Following informed consent, fresh platelet rich plasma (PRP) and, in some cases, buffy coat platelets (the standard of care in Canada and Western Europe) were obtained from volunteer donors or the Canadian Blood Services Network Centre for Applied Development (NetCAD) Laboratory (Vancouver, BC). PRP samples are similar to the platelet preparations used clinically and have an advantage of a lower level of manipulation (*e.g.* centrifugation) than buffy coat or apheresis platelets. Care was taken to assure adequate representation of males and females and no individuals were excluded on

the basis of age (within acceptable age range of donation of 17–71) or race.

present on resting platelets that modulates cell adhesion and migration.

Control and PEGylated mini-units (approximately 50 ml/unit; ~500 × 109

of platelets were stored at 4 or 22°C with agitation per Canadian Blood Services standard operating procedures. Storage at −80°C was done separately in the sample

/L)

PEGylation device to maintain a constant platelet:polymer ratio **(Figure 2)** in a micromixing chamber to assure uniform polymer grafting [50, 52, 56, 71]. Platelets were modified with monofunctional (*i.e.* one binding site per chain) mPEG-succinimidyl propionate (SPA-mPEG). Previous studies (not shown) within our laboratories determined that the optimal molecular weights for the PEGylation of platelets were 2–5 kDa which were used in these studies. For comparison of the effects of different linker chemistry, some studies simultaneously examined mPEG-benzotriazolyl carbonate (BTC-mPEG). Both SPA- and BTC-mPEG (Laysan Bio, Inc., Arab, AL, USA) react with protein lysine residues and covalently attach via formation of a stable amide bond (**Figure 2C**). To demonstrate the efficacy of the polymermediated grafting, the mPEG-mediated immunocamouflage of platelet CD9 was assessed by flow cytometry. CD9 is a constitutive tetraspan membrane glycoprotein

Based on our previous studies, platelets were PEGylated using a semi-automated

**32**

*Production of mPEG-platelets and the SVAmPEG reaction scheme and grafting efficacy. Panel A: semiautomated PEGylation device allowing for control of the mPEG:platelet ratio for uniform grafting levels [71]. Panels B and C: the structure (B) and reaction scheme (C) for activated SVA-mPEG. The SVA-linker chemistry forms a stable amide bond with platelet membrane proteins. Panel D: Fluorescein-conjugated SVAmPEG demonstrated that platelets were uniformly modified using our semi-automated methodology.*

mini-unit blood banking bags. Storage was done for up to 12 days under the prescribed conditions (note: normal storage at 22°C is only allowed for 7 days). Storage bags were sampled aseptically in biosafety cabinets; washing and lysis procedures were performed as described previously [80–82]. Platelet counts were determined using an Advia 120 Hematology Analyzer (Bayer Inc., Toronto, Canada).

## **5. Effect of 4°C storage on mPEG-platelets**

The covalent grafting of mPEG to PRP platelets resulted in the efficient immunocamouflage of CD9 (**Figure 3A**). As demonstrated in **Figure 3A**, virtually 100% of control platelets were CD9<sup>+</sup> , while the mPEG grafting to the platelets exhibited dose effect on the immunocamouflage of CD9. More importantly, the grafted polymer significantly decreased the aggregation of human platelets at 4°C. As microscopically demonstrated in **Figure 3B**, temperature exerted a significant effect on the morphology and microaggregation of control PRP preparations. As anticipated, minimal differences were observed in the control platelets at 37° (*i.e. in vivo* conditions) versus 22°C (normal *in vitro* storage temperature). Importantly, PEGylation with either SCmPEG5000 or BTCmPEG5000 yielded platelets with comparable morphology to the control cells at 37 and 22°C. However, upon thermal transition from 22 to 4°C, control platelets were observed to form significant microaggregates characteristic of the CSL. In stark contrast, neither the SCmPEG5000 nor BTCmPEG5000 modified platelets exhibited any significant microaggregation consequent to the mPEG-mediated inhibition of cell:cell interaction [66, 68]. Morphological analysis of the SCmPEG5000 and BTCmPEG5000 modified platelets suggested that SCmPEG5000 better prevented cold induced shape change relative to the BTC polymer resulting in the SC-linker chemistry being further explored.

The mPEG-mediated inhibition of cold-induced platelet aggregation was also not a short term effect. As demonstrated in **Figure 4**, unmodified control platelets demonstrated significant shape change, microaggregation, and a dramatic (~30%) decrease in platelet count. In contrast, minimal microaggregation was noted in the PEGylated samples following 12 days storage at 4°C. PEGylated platelets also retained a more discoid shape (though some pseudopod formation was noted). Due to the inhibition of microaggregation and inhibition of activation induced shape change, the mPEG-grafted platelets also resulted in a significantly improved platelet count.

Importantly, PEGylated platelets were functionally normal as evidenced by their *in vitro* aggregation response to thrombin. As shown in **Figure 5A**, phase contrast microscopy of washed control and PEGylated platelets resuspended in normal plasma both maintained a smooth, resting morphology. However, in response to 2 IU/mL thrombin activation (**Figure 5B**), both control (a) and PEGylated (b; 10 mM SCmPEG-5000) platelets fully aggregated at 37°C (1000 rpm stir speed) in an aggregometer (Chronolog, Havertown, PA, USA). This normal aggregation of PEGylated platelets occurred despite the significant immunocamouflage (see below) of the platelet membrane surface. Moreover, as shown in **Figure 5C**, phase contrast microscopy demonstrated that control and PEGylated platelets form microscopically very similar thrombin-induced clots. Interestingly, if PEGylated platelets were suspended in PEGylated plasma essentially no *in vitro* aggregation was observed, likely due to the PEGylation of plasma proteins involved in

## **Figure 3.**

*Immunocamouflage of platelets by grafted mPEG. Panel A: CD9 is effectively camouflaged by grafted 5 kDa polymers of SC- and BTC-activated mPEG. The efficacy of immunocamouflage was a function of mPEG grafting concentration. Panel B: While both SC- and BTC-mPEG demonstrated similar efficiency in camouflaging CD9, photomicrographs showed that the SCmPEG better preserved platelet morphology at 4°C. SCmPEG was consequently used for all further studies.*

## **Figure 4.**

*PEGylation inhibited 4°C cold-induced platelets aggregation and shape change. The improved viability of the cells is accompanied by maintenance of the pre-storage platelet count (day 0).*

**35**

**Figure 5.**

*response to ADP, AA, and thrombin activation.*

*Cryoprotection of Platelets by Grafted Polymers DOI: http://dx.doi.org/10.5772/intechopen.89272*

clot formation. However, these data indicated that PEGylated platelets would be functional when transfused into an actively bleeding patient regardless of whether they were stored in the presence of PEGylated plasma, normal plasma or a platelet storage solution as their functionality was restored in the presence of normal plasma. Thromboelastographic (Haemonetics, Braintree, MA) analysis of control and PEGylated platelets further demonstrated the normality of polymer modified platelets in response to multiple platelet agonists. As noted in **Figure 5D**, control and PEGylated platelets demonstrated virtually identical results when exposed to adenosine diphosphate (ADP), arachidonic acid (AA) or thrombin activation indicating that clot formation should not be adversely affected by PEGylation especially since in most circumstances PEGylated donor platelets will represent

*PEGylated platelets are functional and aggregate* in vitro *in response to agonists. Panel A: Phase contrast microscopy of control and PEGylated PRP platelets in plasma demonstrate that both populations maintain a smooth, resting morphology. Panel B: Aggregometer analysis of control (c) and PEGylated (p) platelets demonstrate normal responses to 2 IU/mL thrombin (37°C; 1000 rpm). Panel C: Control and PEGylated PRP platelets form microscopically very similar thrombin-induced clots. Panel D: PEGylation did not affect platelet thromboelastography (TEG) as denoted by the virtually identical TEG tracings. These results demonstrated that PEGylated platelets participated normally in the in vitro clotting assay. Platelet mapping with TEG determines total platelet function. The two symmetric arms show the same results. Shown are representative responses of control (c; black lines) and PEGylated (red lines; SCmPEG5000) platelets at rest (baseline) and in* 

Clinically, visual inspection for 'swirl' may be the only pre-transfusion 'quality' test of the platelet unit—though even this is rarely done. The swirl test is a

~50% or, most typically, much less of the platelets in a clot.

*Cryoprotection of Platelets by Grafted Polymers DOI: http://dx.doi.org/10.5772/intechopen.89272*

*Cryopreservation - Current Advances and Evaluations*

*4°C. SCmPEG was consequently used for all further studies.*

Importantly, PEGylated platelets were functionally normal as evidenced by their *in vitro* aggregation response to thrombin. As shown in **Figure 5A**, phase contrast microscopy of washed control and PEGylated platelets resuspended in normal plasma both maintained a smooth, resting morphology. However, in response to 2 IU/mL thrombin activation (**Figure 5B**), both control (a) and PEGylated (b; 10 mM SCmPEG-5000) platelets fully aggregated at 37°C (1000 rpm stir speed) in an aggregometer (Chronolog, Havertown, PA, USA). This normal aggregation of PEGylated platelets occurred despite the significant immunocamouflage (see below) of the platelet membrane surface. Moreover, as shown in **Figure 5C**, phase contrast microscopy demonstrated that control and PEGylated platelets form microscopically very similar thrombin-induced clots. Interestingly, if PEGylated platelets were suspended in PEGylated plasma essentially no *in vitro* aggregation was observed, likely due to the PEGylation of plasma proteins involved in

*PEGylation inhibited 4°C cold-induced platelets aggregation and shape change. The improved viability of the* 

*Immunocamouflage of platelets by grafted mPEG. Panel A: CD9 is effectively camouflaged by grafted 5 kDa polymers of SC- and BTC-activated mPEG. The efficacy of immunocamouflage was a function of mPEG grafting concentration. Panel B: While both SC- and BTC-mPEG demonstrated similar efficiency in camouflaging CD9, photomicrographs showed that the SCmPEG better preserved platelet morphology at* 

*cells is accompanied by maintenance of the pre-storage platelet count (day 0).*

**34**

**Figure 4.**

**Figure 3.**

### **Figure 5.**

*PEGylated platelets are functional and aggregate* in vitro *in response to agonists. Panel A: Phase contrast microscopy of control and PEGylated PRP platelets in plasma demonstrate that both populations maintain a smooth, resting morphology. Panel B: Aggregometer analysis of control (c) and PEGylated (p) platelets demonstrate normal responses to 2 IU/mL thrombin (37°C; 1000 rpm). Panel C: Control and PEGylated PRP platelets form microscopically very similar thrombin-induced clots. Panel D: PEGylation did not affect platelet thromboelastography (TEG) as denoted by the virtually identical TEG tracings. These results demonstrated that PEGylated platelets participated normally in the in vitro clotting assay. Platelet mapping with TEG determines total platelet function. The two symmetric arms show the same results. Shown are representative responses of control (c; black lines) and PEGylated (red lines; SCmPEG5000) platelets at rest (baseline) and in response to ADP, AA, and thrombin activation.*

clot formation. However, these data indicated that PEGylated platelets would be functional when transfused into an actively bleeding patient regardless of whether they were stored in the presence of PEGylated plasma, normal plasma or a platelet storage solution as their functionality was restored in the presence of normal plasma. Thromboelastographic (Haemonetics, Braintree, MA) analysis of control and PEGylated platelets further demonstrated the normality of polymer modified platelets in response to multiple platelet agonists. As noted in **Figure 5D**, control and PEGylated platelets demonstrated virtually identical results when exposed to adenosine diphosphate (ADP), arachidonic acid (AA) or thrombin activation indicating that clot formation should not be adversely affected by PEGylation especially since in most circumstances PEGylated donor platelets will represent ~50% or, most typically, much less of the platelets in a clot.

Clinically, visual inspection for 'swirl' may be the only pre-transfusion 'quality' test of the platelet unit—though even this is rarely done. The swirl test is a

noninvasive method that literally works by swirling the bag and looking for light diffraction (*i.e.* refractiveness) [83–86]. Due to the discoid shape of resting (unactivated) platelets, light is diffracted creating a cloud- or swirl-like appearance of the bag. Platelet activation causes a disc to sphere morphology change where upon orientation dependent changes in light diffraction are no longer observed. A dull platelet bag is deemed 'activated' while a refractive bag is 'resting'. Unsurprisingly, despite the low cost (*i.e.* free) of the swirl test, it actually tells very little as to the quality of the platelet unit. Over the last few years, a new technology has been developed to quantitatively measure platelet quality using dynamic light scattering. The ThromboLUX (LightIntegra Technology, Vancouver, BC) quantitatively measures platelet morphology (shape change) and temperature response and provides a quantitative replacement to the qualitative and subjective 'platelet swirl' [62, 83–85, 87–92]. Mechanistically the ThromboLUX utilizes dynamic light scattering to examine a small volume (~30 μl) of platelets to quantitatively assess platelet size and morphological changes arising from temperature cycling (37 to 20 to 37°C). The ThomboLUX generates a Dynamic Light Scattering (DLS) value that correlates with platelet activation status. Moreover, the ThromboLUX is capable of quantitating the number of platelet-derived microparticles and evidence of microbial contamination. The ThromboLUX technology has been clinically validated and provides a correlation between the DLS score and a patients corrected count increment at 24 hours (CCI24) post transfusion [84]. Shown in **Figure 6A** is the ThromboLUX

## **Figure 6.**

*ThromboLUX dynamic light scattering analysis of platelets following 6 days storage. Panel A: Control platelets stored at 22°C. panel B: Control platelets stored at 4°C. panel C: ScmPEG5000 (10 mM) platelets stored at 4°C. As shown, mPEG grafting resulted in significant cryoprotection. The Y-axis is particle count while the x-axis reflects the hydrodynamic radius distribution. All samples were prepared from the same donor platelet unit.*

**37**

**Figure 7.**

*Cryoprotection of Platelets by Grafted Polymers DOI: http://dx.doi.org/10.5772/intechopen.89272*

and minimal microparticle formation (**Figure 6C**).

**6. Effect of −80°C storage on mPEG-platelets**

profile of control platelets stored 6 days at 22°C. However, cold storage of the same platelet preparation resulted in a dramatic shift of the platelet peak (**Figure 6B**) with the appearance of microparticles, smaller platelets and platelet aggregates. In contrast, cold stored (6 days) PEGylated platelets (10 mM, 5 kDa) yielded a DLS profile similar to the 22°C stored platelets with no evidence of aggregate formation

To further assess the cryoprotective effects of the grafted mPEG polymer, freezing studies were conducted on the control and SCmPEG platelets. While previous work on PEG as a cryoprotectant utilized a soluble form, work with PEG and other cryoprotectants (DMSO and Trehalose) demonstrated that the primary site of protection was at the level of the cell membrane [72–79]. As shown in **Figure 7**, following 12 days storage at −80°C, covalently bound SCmPEG provided significant cryoprotection as reflected by both platelet morphology and improved cell counts. This finding is in stark contrast to control platelets which exhibited significant fragmentation and dramatically reduced cell counts post storage and thawing. The covalent grafted mPEG exerted additional benefits post thaw. As shown in **Figure 8**, SCmPEG-grafted platelets exhibited improved morphology, and less fragmentation, immediately post-thaw when compared to control cells. Indeed, the grafted polymer provided comparable (or better) cryoprotection than DMSO. Moreover, following washing and re-concentration of the freeze-thaw

*PEGylation inhibited −80°C cold-induced platelets aggregation and shape change. The improved viability of the cells is accompanied by significantly improved maintenance of the pre-storage platelet count (day 0).*

*Cryoprotection of Platelets by Grafted Polymers DOI: http://dx.doi.org/10.5772/intechopen.89272*

*Cryopreservation - Current Advances and Evaluations*

noninvasive method that literally works by swirling the bag and looking for light diffraction (*i.e.* refractiveness) [83–86]. Due to the discoid shape of resting (unactivated) platelets, light is diffracted creating a cloud- or swirl-like appearance of the bag. Platelet activation causes a disc to sphere morphology change where upon orientation dependent changes in light diffraction are no longer observed. A dull platelet bag is deemed 'activated' while a refractive bag is 'resting'. Unsurprisingly, despite the low cost (*i.e.* free) of the swirl test, it actually tells very little as to the quality of the platelet unit. Over the last few years, a new technology has been developed to quantitatively measure platelet quality using dynamic light scattering. The ThromboLUX (LightIntegra Technology, Vancouver, BC) quantitatively measures platelet morphology (shape change) and temperature response and provides a quantitative replacement to the qualitative and subjective 'platelet swirl' [62, 83–85, 87–92]. Mechanistically the ThromboLUX utilizes dynamic light scattering to examine a small volume (~30 μl) of platelets to quantitatively assess platelet size and morphological changes arising from temperature cycling (37 to 20 to 37°C). The ThomboLUX generates a Dynamic Light Scattering (DLS) value that correlates with platelet activation status. Moreover, the ThromboLUX is capable of quantitating the number of platelet-derived microparticles and evidence of microbial contamination. The ThromboLUX technology has been clinically validated and provides a correlation between the DLS score and a patients corrected count increment at 24 hours (CCI24) post transfusion [84]. Shown in **Figure 6A** is the ThromboLUX

**36**

*unit.*

**Figure 6.**

*ThromboLUX dynamic light scattering analysis of platelets following 6 days storage. Panel A: Control platelets stored at 22°C. panel B: Control platelets stored at 4°C. panel C: ScmPEG5000 (10 mM) platelets stored at 4°C. As shown, mPEG grafting resulted in significant cryoprotection. The Y-axis is particle count while the x-axis reflects the hydrodynamic radius distribution. All samples were prepared from the same donor platelet* 

profile of control platelets stored 6 days at 22°C. However, cold storage of the same platelet preparation resulted in a dramatic shift of the platelet peak (**Figure 6B**) with the appearance of microparticles, smaller platelets and platelet aggregates. In contrast, cold stored (6 days) PEGylated platelets (10 mM, 5 kDa) yielded a DLS profile similar to the 22°C stored platelets with no evidence of aggregate formation and minimal microparticle formation (**Figure 6C**).

## **6. Effect of −80°C storage on mPEG-platelets**

To further assess the cryoprotective effects of the grafted mPEG polymer, freezing studies were conducted on the control and SCmPEG platelets. While previous work on PEG as a cryoprotectant utilized a soluble form, work with PEG and other cryoprotectants (DMSO and Trehalose) demonstrated that the primary site of protection was at the level of the cell membrane [72–79]. As shown in **Figure 7**, following 12 days storage at −80°C, covalently bound SCmPEG provided significant cryoprotection as reflected by both platelet morphology and improved cell counts. This finding is in stark contrast to control platelets which exhibited significant fragmentation and dramatically reduced cell counts post storage and thawing.

The covalent grafted mPEG exerted additional benefits post thaw. As shown in **Figure 8**, SCmPEG-grafted platelets exhibited improved morphology, and less fragmentation, immediately post-thaw when compared to control cells. Indeed, the grafted polymer provided comparable (or better) cryoprotection than DMSO. Moreover, following washing and re-concentration of the freeze-thaw

## **Figure 7.**

*PEGylation inhibited −80°C cold-induced platelets aggregation and shape change. The improved viability of the cells is accompanied by significantly improved maintenance of the pre-storage platelet count (day 0).*

### **Figure 8.**

*PEGylation gives rise to significant cryoprotection from freeze-thaw injury. As shown, after 12 days of storage at −80°C, unmodified control platelets exhibited significant platelet destruction, loss of morphology, and a significant (p < 0.001) decrease in platelet count. In contrast, subsequent to freeze-thaw, PEGylated platelets demonstrated normal discoid morphology and few detectable microaggregates and were very comparable to platelets cryopreserved with DMSO, the standard cryoprotectant for frozen RBC and platelets. Moreover, unlike control platelets, the PEGylated sample did not form microaggregates when incubated at 22°C.*

### **Figure 9.**

*Post storage at −80°C, the aggregation of control and SCmPEG platelets was assessed in response to thrombin (2 IU/mL). As shown, thawed control platelets exhibited limited aggregation (~40% light transmittance) after 8 minutes. In contrast, the PEGylated platelets demonstrated robust aggregation (100% light transmittance at ~4 minutes). Shown are representative responses of control (black lines) and PEGylated (red lines; SCmPEG5000) platelets.*

**39**

for 12 hours.

*Cryoprotection of Platelets by Grafted Polymers DOI: http://dx.doi.org/10.5772/intechopen.89272*

**Figure 5B**).

**7. Discussion**

are clinically suitable are ongoing.

control and SCmPEG platelets, control platelets demonstrated significant aggregation when stored at 22°C overnight (12 hours). In contrast, the SCmPEG-platelets

While the maintenance of morphology and platelet numbers post −80 storage was promising, the key question was whether these platelets were functional. To assess platelet function, thrombin (2 IU/ml) induced aggregation was assessed. As shown in **Figure 9**, thawed control platelets exhibited a poor response to thrombin (see **Figure 5B** for a normal response) as demonstrated by very limited aggregation. Moreover, the aggregation of the control platelets was very slow as seen by the slope of the aggregation curve. In contrast, the thawed SCmPEG platelets demonstrated significant, and rapid, thrombin mediated aggregation. Indeed, near maximal aggregation was achieved within approximately 3 minutes and very closely resembled the thrombin activation curves of fresh control and SCmPEG PRP (see

Platelet transfusions are a critical component in the treatment of both traumatic acute injury and a number of chronic diseases. However, unlike RBC which are stored at 4°C, platelets are stored at 22–24°C (room temperature) due to the induction of the CSL at temperatures below ~18°C. While the CSL encompasses a multitude of biophysical and biochemical changes, perhaps the most apparent effect is the production of platelet aggregates. To prevent the CSL, blood services worldwide have successfully stored platelets at 22°C. However, warm storage has its own risks as it greatly increases the risk for microbial growth limiting the safe storage of platelets to only 5–7 days (versus 42 days for RBC) and the outdating of a significant number of donor units. Consequent to the short shelf life of platelets, blood services face chronic shortages of these life-saving cells. To overcome both the risk of microbial contamination and the constrained supplies of platelets, renewed research into attenuating the CSL and/or determining where cold stored platelets

To circumvent the microbial risk, and improve platelet inventory, our research has examined the potential use of cold stored, mPEG-grafted, platelets. As demonstrated by our *in vitro* experimental findings, the covalent grafting of mPEG to donor platelets significantly reduced the severity of the 4°C CSL while maintaining normal haemostatic function. This was evidenced by the maintenance of 'normal' platelet morphology, the lack of microaggregation, and normal platelet activation by thrombin and normal aggregation as determined by thromboelastography and aggregation studies. Moreover, due to the cryoprotective effects of the grafted mPEG, polymer-modified platelets could be stored at −80°C and thawed and still retained normal morphology and haemostatic function. In contrast, the freezing of normal platelets in the absence of a cryoprotectant (*e.g.* DMSO) resulted in significant cellular damage resulting in vastly reduced recovery, loss of haemostatic function and subsequent microaggregation when incubated at room temperature

Interestingly, while not a focus of this chapter, polymer size (*e.g.* 2, 5 or 20 kDa) was an important factor when considering the cryoprotection of platelets while maintaining the unique functions of the platelet. In contrast to RBC and WBC in which longer chain polymers (*e.g.* 20 kDa) were optimal, short chain polymers (*e.g.* 2–5 kDa) were optimal for platelet cryopreservation. However, the disparity between RBC and WBC relative to platelets is not as unanticipated as it appears.

demonstrated no aggregation over the same 12 hour time frame.

## *Cryoprotection of Platelets by Grafted Polymers DOI: http://dx.doi.org/10.5772/intechopen.89272*

control and SCmPEG platelets, control platelets demonstrated significant aggregation when stored at 22°C overnight (12 hours). In contrast, the SCmPEG-platelets demonstrated no aggregation over the same 12 hour time frame.

While the maintenance of morphology and platelet numbers post −80 storage was promising, the key question was whether these platelets were functional. To assess platelet function, thrombin (2 IU/ml) induced aggregation was assessed. As shown in **Figure 9**, thawed control platelets exhibited a poor response to thrombin (see **Figure 5B** for a normal response) as demonstrated by very limited aggregation. Moreover, the aggregation of the control platelets was very slow as seen by the slope of the aggregation curve. In contrast, the thawed SCmPEG platelets demonstrated significant, and rapid, thrombin mediated aggregation. Indeed, near maximal aggregation was achieved within approximately 3 minutes and very closely resembled the thrombin activation curves of fresh control and SCmPEG PRP (see **Figure 5B**).

## **7. Discussion**

*Cryopreservation - Current Advances and Evaluations*

**38**

**Figure 9.**

**Figure 8.**

*SCmPEG5000) platelets.*

*Post storage at −80°C, the aggregation of control and SCmPEG platelets was assessed in response to thrombin (2 IU/mL). As shown, thawed control platelets exhibited limited aggregation (~40% light transmittance) after 8 minutes. In contrast, the PEGylated platelets demonstrated robust aggregation (100% light transmittance at ~4 minutes). Shown are representative responses of control (black lines) and PEGylated (red lines;* 

*PEGylation gives rise to significant cryoprotection from freeze-thaw injury. As shown, after 12 days of storage at −80°C, unmodified control platelets exhibited significant platelet destruction, loss of morphology, and a significant (p < 0.001) decrease in platelet count. In contrast, subsequent to freeze-thaw, PEGylated platelets demonstrated normal discoid morphology and few detectable microaggregates and were very comparable to platelets cryopreserved with DMSO, the standard cryoprotectant for frozen RBC and platelets. Moreover, unlike control platelets, the PEGylated sample did not form microaggregates when incubated at 22°C.*

Platelet transfusions are a critical component in the treatment of both traumatic acute injury and a number of chronic diseases. However, unlike RBC which are stored at 4°C, platelets are stored at 22–24°C (room temperature) due to the induction of the CSL at temperatures below ~18°C. While the CSL encompasses a multitude of biophysical and biochemical changes, perhaps the most apparent effect is the production of platelet aggregates. To prevent the CSL, blood services worldwide have successfully stored platelets at 22°C. However, warm storage has its own risks as it greatly increases the risk for microbial growth limiting the safe storage of platelets to only 5–7 days (versus 42 days for RBC) and the outdating of a significant number of donor units. Consequent to the short shelf life of platelets, blood services face chronic shortages of these life-saving cells. To overcome both the risk of microbial contamination and the constrained supplies of platelets, renewed research into attenuating the CSL and/or determining where cold stored platelets are clinically suitable are ongoing.

To circumvent the microbial risk, and improve platelet inventory, our research has examined the potential use of cold stored, mPEG-grafted, platelets. As demonstrated by our *in vitro* experimental findings, the covalent grafting of mPEG to donor platelets significantly reduced the severity of the 4°C CSL while maintaining normal haemostatic function. This was evidenced by the maintenance of 'normal' platelet morphology, the lack of microaggregation, and normal platelet activation by thrombin and normal aggregation as determined by thromboelastography and aggregation studies. Moreover, due to the cryoprotective effects of the grafted mPEG, polymer-modified platelets could be stored at −80°C and thawed and still retained normal morphology and haemostatic function. In contrast, the freezing of normal platelets in the absence of a cryoprotectant (*e.g.* DMSO) resulted in significant cellular damage resulting in vastly reduced recovery, loss of haemostatic function and subsequent microaggregation when incubated at room temperature for 12 hours.

Interestingly, while not a focus of this chapter, polymer size (*e.g.* 2, 5 or 20 kDa) was an important factor when considering the cryoprotection of platelets while maintaining the unique functions of the platelet. In contrast to RBC and WBC in which longer chain polymers (*e.g.* 20 kDa) were optimal, short chain polymers (*e.g.* 2–5 kDa) were optimal for platelet cryopreservation. However, the disparity between RBC and WBC relative to platelets is not as unanticipated as it appears.

The goal of RBC and WBC PEGylation is to induce immunocamouflage (*i.e.* prevent immune recognition) and, especially for WBC, prevent cell:cell communication (*e.g.* allorecognition). Clearly for platelets, cell:cell interaction is crucial for their haemostatic function. Hence, the primary goal of platelet PEGylation is to prevent thermal injury to the membrane while maintaining normal platelet function. While all sizes of the grafted polymer could prevent cryogenic injury, long chain polymers inhibited platelet activation and their subsequent aggregation. In contrast, the short 2–5 kDa polymers provided adequate cryoprotective effects while allowing for normal agonist-driven activation.

## **8. Conclusions**

PEGylation of donor platelets with short chain (2–5 kDa) mPEG effectively prevent the overt morphological changes arising from the CSL. Moreover, the polymer-grafted platelets retained their normal haemostatic function following both cold storage (4°C) and freezing (−80°C) as evidenced by thromboelastography and aggregation studies. Importantly, cold storage of platelets would improve transfusion safety as it would diminish the risk of microbial growth in a blood product destined for use in at risk patients. Also of potential clinical and economic importance was the finding that mPEG-grafted platelets withstood freezing in the absence of other cryoprotectants such as DMSO. The use of frozen platelets, requiring no DMSO removal step, could expand the availability of platelet transfusions to geographic regions in which they are not currently available or where donor recruitment or production facilities do not exist. The successful implementation of this technology for the cold storage of platelets would be of significant benefit to transfusion recipients by increasing the availability of platelets for transfusion.

## **Acknowledgements**

This work was supported by grants from the Canadian Blood Services (MDS; EMS and MDS) and Health Canada (MDS). The views expressed herein do not necessarily represent the view of the federal government of Canada. We thank the Canada Foundation for Innovation and the Michael Smith Foundation for Health Research for infrastructure funding at the University of British Columbia Centre for Blood Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

## **Conflict of interest**

The Canadian Blood Services (Ottawa, ON, Canada) has patents relating to the cold storage of platelets [48, 49]. MDS, NN and EMS are inventors cited on said patents.

**41**

**Author details**

Canada

Mark D. Scott1,2,3\*, Nobu Nakane1

Columbia, Vancouver, BC, Canada

provided the original work is properly cited.

1 Canadian Blood Services, Ottawa, ON, Canada

\*Address all correspondence to: mdscott@mail.ubc.ca

and Elisabeth Maurer-Spurej1,2,3

2 The Centre for Blood Research, University of British Columbia, Vancouver, BC,

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

3 Department of Pathology and Laboratory Medicine, University of British

*Cryoprotection of Platelets by Grafted Polymers DOI: http://dx.doi.org/10.5772/intechopen.89272* *Cryoprotection of Platelets by Grafted Polymers DOI: http://dx.doi.org/10.5772/intechopen.89272*

*Cryopreservation - Current Advances and Evaluations*

normal agonist-driven activation.

**8. Conclusions**

**Acknowledgements**

**Conflict of interest**

The goal of RBC and WBC PEGylation is to induce immunocamouflage (*i.e.* prevent immune recognition) and, especially for WBC, prevent cell:cell communication (*e.g.* allorecognition). Clearly for platelets, cell:cell interaction is crucial for their haemostatic function. Hence, the primary goal of platelet PEGylation is to prevent thermal injury to the membrane while maintaining normal platelet function. While all sizes of the grafted polymer could prevent cryogenic injury, long chain polymers inhibited platelet activation and their subsequent aggregation. In contrast, the short 2–5 kDa polymers provided adequate cryoprotective effects while allowing for

PEGylation of donor platelets with short chain (2–5 kDa) mPEG effectively prevent the overt morphological changes arising from the CSL. Moreover, the polymer-grafted platelets retained their normal haemostatic function following both cold storage (4°C) and freezing (−80°C) as evidenced by thromboelastography and aggregation studies. Importantly, cold storage of platelets would improve transfusion safety as it would diminish the risk of microbial growth in a blood product destined for use in at risk patients. Also of potential clinical and economic importance was the finding that mPEG-grafted platelets withstood freezing in the absence of other cryoprotectants such as DMSO. The use of frozen platelets, requiring no DMSO removal step, could expand the availability of platelet transfusions to geographic regions in which they are not currently available or where donor recruitment or production facilities do not exist. The successful implementation of this technology for the cold storage of platelets would be of significant benefit to transfusion recipients by increasing the availability of platelets for transfusion.

This work was supported by grants from the Canadian Blood Services (MDS; EMS and MDS) and Health Canada (MDS). The views expressed herein do not necessarily represent the view of the federal government of Canada. We thank the Canada Foundation for Innovation and the Michael Smith Foundation for Health Research for infrastructure funding at the University of British Columbia Centre for Blood Research. The funders had no role in study design, data collection and

The Canadian Blood Services (Ottawa, ON, Canada) has patents relating to the cold storage of platelets [48, 49]. MDS, NN and EMS are inventors cited on said

analysis, decision to publish, or preparation of the manuscript.

**40**

patents.

## **Author details**

Mark D. Scott1,2,3\*, Nobu Nakane1 and Elisabeth Maurer-Spurej1,2,3

1 Canadian Blood Services, Ottawa, ON, Canada

2 The Centre for Blood Research, University of British Columbia, Vancouver, BC, Canada

3 Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada

\*Address all correspondence to: mdscott@mail.ubc.ca

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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asheducation-2003.1.575

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safety of platelet transfusions. Transfusion Medicine Reviews. 2004;**18**:11-24. DOI: 10.1016/j.

tmrv.2003.10.002

1995;**24**:163-170. DOI: 10.3109/08820139509062770 *Cryoprotection of Platelets by Grafted Polymers DOI: http://dx.doi.org/10.5772/intechopen.89272*

Mayadas TN, et al. The clearance mechanism of chilled blood platelets. Cell. 2003;**112**:87-97. DOI: 10.1016/ S0092-8674(02)01253-9

[20] Wagner SJ, Friedman LI, Dodd RY. Transfusion-associated bacterial sepsis. Clinical Microbiology Reviews. 1994;**7**:290-302. DOI: 10.1128/ CMR.7.3.290

[21] Blajchman MA. Bacterial contamination of blood products and the value of pre-transfusion testing. Immunological Investigations. 1995;**24**:163-170. DOI: 10.3109/08820139509062770

[22] Dumont LJ, AuBuchon JP, Whitley P, Herschel LH, Johnson A, McNeil D, et al. Seven-day storage of single-donor platelets: Recovery and survival in an autologous transfusion study. Transfusion. 2002;**42**:847-854. DOI: 10.1046/j.1537-2995.2002.00147.x

[23] Hillyer CD, Josephson CD, Blajchman MA, Vostal JG, Epstein JS, Goodman JL. Bacterial contamination of blood components: Risks, strategies, and regulation: Joint ASH and AABB educational session in transfusion medicine. Hematology. American Society of Hematology. Education Program. 2003;**2003**:575-589. DOI: 10.1182/ asheducation-2003.1.575

[24] Benjamin RJ, Wagner SJ. The residual risk of sepsis: Modeling the effect of concentration on bacterial detection in two-bottle culture systems and an estimation of false-negative culture rates. Transfusion. 2007;**47**:1381-1389. DOI: 10.1111/j.1537-2995.2007.01326.x

[25] Blajchman MA, Goldman M, Baeza F. Improving the bacteriological safety of platelet transfusions. Transfusion Medicine Reviews. 2004;**18**:11-24. DOI: 10.1016/j. tmrv.2003.10.002

[26] Blajchman MA, Beckers EA, Dickmeiss E, Lin L, Moore G, Muylle L. Bacterial detection of platelets: Current problems and possible resolutions. Transfusion Medicine Reviews. 2005;**19**:259-272. DOI: 10.1016/j.tmrv.2005.05.002

[27] Wandall HH, Hoffmeister KM, Sorensen AL, Rumjantseva V, Clausen H, Hartwig JH, et al. Galactosylation does not prevent the rapid clearance of long-term, 4 degrees C-stored platelets. Blood. 2008;**111**:3249-3256. DOI: 10.1182/ blood-2007-06-097295

[28] Milford EM, Reade MC. Comprehensive review of platelet storage methods for use in the treatment of active hemorrhage. Transfusion. 2016;**56**(Suppl 2):S140-S148. DOI: 10.1111/trf.13504

[29] Johnson L, Tan S, Wood B, Davis A, Marks DC. Refrigeration and cryopreservation of platelets differentially affect platelet metabolism and function: A comparison with conventional platelet storage conditions. Transfusion. 2016;**56**:1807-1818. DOI: 10.1111/trf.13630

[30] Getz TM, Montgomery RK, Bynum JA, Aden JK, Pidcoke HF, Cap AP. Storage of platelets at 4°C in platelet additive solutions prevents aggregate formation and preserves platelet functional responses. Transfusion. 2016;**56**:1320-1328. DOI: 10.1111/trf.13511

[31] Spinella PC, Cap AP. Whole blood: Back to the future. Current Opinion in Hematology. 2016;**23**:536-542. DOI: 10.1097/MOH.0000000000000284

[32] Stubbs JR, Tran SA, Emery RL, Hammel SA, Haugen AL, Zielinski MD, et al. Cold platelets for traumaassociated bleeding: Regulatory approval, accreditation approval, and practice implementation-just the "tip of

**42**

*Cryopreservation - Current Advances and Evaluations*

[10] Murphy S, Sayar SN, Gardner FH. Storage of platelet concentrates at 22 degrees C. Blood. 1970;**35**:549-557

Maintenance of platelet viability and functional integrity during storage. Vox Sanguinis. 1971;**20**:427-428. DOI: 10.1111/j.1423-0410.1971.tb01814.x

[12] Murphy S, Gardner FH. Platelet storage at 22 degrees C; metabolic, morphologic, and functional studies. The Journal of Clinical Investigation. 1971;**50**:370-377. DOI: 10.1172/

[13] Becker GA, Tuccelli M, Kunicki T, Chalos MK, Aster RH. Studies of platelet

[14] Slichter SJ, Harker LA. Preparation and storage of platelet concentrates.

[16] Slichter SJ. Preservation of platelet viability and function during storage of concentrates. Progress in Clinical and Biological Research. 1978;**28**:83-100

[17] Winokur R, Hartwig JH. Mechanism of shape change in chilled human platelets. Blood. 1995;**85**:1796-1804

[18] Hoffmeister KM, Josefsson EC, Isaac NA, Clausen H, Hartwig JH, Stossel TP. Glycosylation restores survival of chilled blood platelets. Science. 2003;**301**:1531-1534. DOI:

[19] Hoffmeister KM, Felbinger TW, Falet H, Denis CV, Bergmeier W,

10.1126/science.1085322

concentrates stored at 22 C and 4 C. Transfusion. 1973;**13**:61-68. DOI: 10.1111/j.1537-2995.1973.tb05442.x

[15] Holme S, Vaidja K, Murphy S. Platelet storage at 22 degrees C: Effect of type of agitation on morphology, viability, and function in vitro. Blood.

Transfusion. 1976;**16**:8-12

1978;**52**:425-435

[11] Murphy S, Gardner FH.

JCI106504

[1] Slichter SJ. Platelet transfusion therapy. Hematology/Oncology Clinics of North America. 1990;**4**:291-311. DOI:

**References**

10.1016/S0889-8588(18)30517-3

[2] Murphy S, Varma M. Selecting platelets for transfusion of the alloimmunized patient: A review. Immunohematology. 1998;**14**:117-123

[3] Hartwig JH. The platelet: Form and function. Seminars in Hematology. 2006;**43**:S94-S100. DOI: 10.1053/j. seminhematol.2005.11.004

[4] Wohner N. Role of cellular elements in thrombus formation and dissolution. Cardiovascular & Hematological Agents in Medicinal Chemistry. 2008;**6**:224- 228. DOI: 10.2174/187152508784871972

[5] Cobain TJ, Vamvakas EC, Wells A,

demographics of blood use. Transfusion

Titlestad K. A survey of the

Medicine. 2007;**17**:1-15. DOI: 10.1111/j.1365-3148.2006.00709.x

[7] Goldman M, Steele WR, Di Angelantonio E, van den

[6] Stroncek DF, Rebulla P. Platelet transfusions. Lancet. 2007;**370**:427-438. DOI: 10.1016/S0140-6736(07)61198-2

Hurk K, Vassallo RR, Germain M, et al. Biomedical EFSTCBESTI. Comparison of donor and general population demographics over time: A BEST collaborative group study. Transfusion. 2017;**57**:2469-2476. DOI: 10.1111/trf.14307

[8] Murphy S, Gardner FH. Effect of storage temperature on maintenance of platelet viability--deleterious effect of refrigerated storage. The New England Journal of Medicine. 1969;**280**:1094-1098. DOI: 10.1056/

[9] Murphy S, Gardner FH. The effect of temperature on platelet viability. Vox

NEJM196905152802004

Sanguinis. 1969;**17**:22

the iceberg". Transfusion. 2017;**57**: 2836-2844. DOI: 10.1111/trf.14303

[33] Berzuini A, Spreafico M, Prati D. One size doesn't fit all: Should we reconsider the introduction of cold-stored platelets in blood bank inventories. F1000Res. 2017;**6**:95. DOI: 10.12688/f1000research.10363.1

[34] Wu X, Darlington DN, Montgomery RK, Liu B, Keesee JD, Scherer MR, et al. Platelets derived from fresh and cold-stored whole blood participate in clot formation in rats with acute traumatic coagulopathy. British Journal of Haematology. 2017;**179**:802- 810. DOI: 10.1111/bjh.14999

[35] Stolla M, Fitzpatrick L, Gettinger I, Bailey SL, Pellham E, Christoffel T, et al. In vivo viability of extended 4°C-stored autologous apheresis platelets. Transfusion. 2018;**58**:2407-2413. DOI: 10.1111/trf.14833

[36] Ng MSY, Tung JP, Fraser JF. Platelet storage lesions: What more do we know now. Transfusion Medicine Reviews. 2018;**32**:144-154. DOI: 10.1016/j. tmrv.2018.04.001

[37] Humbrecht C, Kientz D, Gachet C. Platelet transfusion: Current challenges. Transfusion Clinique et Biologique. 2018;**25**:151-164. DOI: 10.1016/j.tracli.2018.06.004

[38] Waters L, Cameron M, Padula MP, Marks DC, Johnson L. Refrigeration, cryopreservation and pathogen inactivation: An updated perspective on platelet storage conditions. Vox Sanguinis. 2018;**113**:317-328. DOI: 10.1111/vox.12640

[39] Reddoch-Cardenas KM, Sharma U, Salgado CL, Montgomery RK, Cantu C, Cingoz N, et al. An in vitro pilot study of apheresis platelets collected on Trima Accel system and stored in T-PAS+ solution at refrigeration temperature (1-6°C). Transfusion.

2019;**59**:1789-1798. DOI: 10.1111/ trf.15150

[40] Reddoch-Cardenas KM, Bynum JA, Meledeo MA, Nair PM, Wu X, Darlington DN, et al. Coldstored platelets: A product with function optimized for hemorrhage control. Transfusion and Apheresis Science. 2019;**58**:16-22. DOI: 10.1016/j. transci.2018.12.012

[41] Leeper CM, Yazer MH, Cladis FP, Saladino R, Triulzi DJ, Gaines BA. Cold-stored whole blood platelet function is preserved in injured children with hemorrhagic shock. Journal of Trauma and Acute Care Surgery. 2019;**87**:49-53. DOI: 10.1097/ TA.0000000000002340

[42] Braathen H, Sivertsen J, Lunde THF, Kristoffersen EK, Assmus J, Hervig TA, et al. In vitro quality and platelet function of cold and delayed cold storage of apheresis platelet concentrates in platelet additive solution for 21days. Transfusion. 2019;**58**:2652-2661. DOI: 10.1111/ trf.15356

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[44] Getz TM. Physiology of cold-stored platelets. Transfusion and Apheresis Science. 2019;**58**:12-15. DOI: 10.1016/j. transci.2018.12.011

[45] Ketter PM, Kamucheka R, Arulanandam B, Akers K, Cap AP. Platelet enhancement of bacterial growth during room temperature storage: Mitigation through refrigeration. Transfusion. 2019;**59**:1479-1489. DOI: 10.1111/ trf.15255

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*Cryoprotection of Platelets by Grafted Polymers DOI: http://dx.doi.org/10.5772/intechopen.89272*

*Cryopreservation - Current Advances and Evaluations*

2019;**59**:1789-1798. DOI: 10.1111/

[40] Reddoch-Cardenas KM, Bynum JA, Meledeo MA, Nair PM, Wu X, Darlington DN, et al. Coldstored platelets: A product with function optimized for hemorrhage control. Transfusion and Apheresis Science. 2019;**58**:16-22. DOI: 10.1016/j.

transci.2018.12.012

[41] Leeper CM, Yazer MH, Cladis FP, Saladino R, Triulzi DJ, Gaines BA. Cold-stored whole blood platelet function is preserved in injured children with hemorrhagic shock. Journal of Trauma and Acute Care Surgery. 2019;**87**:49-53. DOI: 10.1097/

TA.0000000000002340

[43] Scorer T, Williams A,

transci.2018.12.011

trf.15255

[45] Ketter PM, Kamucheka R, Arulanandam B, Akers K, Cap AP. Platelet enhancement of bacterial growth during room temperature storage: Mitigation through refrigeration. Transfusion. 2019;**59**:1479-1489. DOI: 10.1111/

Reddoch-Cardenas K, Mumford A. Manufacturing variables and hemostatic function of cold-stored platelets: A systematic review of the literature. Transfusion. 2019;**59**: 2722-2732. DOI: 10.1111/trf.15396

[44] Getz TM. Physiology of cold-stored platelets. Transfusion and Apheresis Science. 2019;**58**:12-15. DOI: 10.1016/j.

trf.15356

[42] Braathen H, Sivertsen J, Lunde THF, Kristoffersen EK, Assmus J, Hervig TA, et al. In vitro quality and platelet function of cold and delayed cold storage of apheresis platelet concentrates in platelet additive solution for 21days. Transfusion. 2019;**58**:2652-2661. DOI: 10.1111/

trf.15150

the iceberg". Transfusion. 2017;**57**: 2836-2844. DOI: 10.1111/trf.14303

Prati D. One size doesn't fit all: Should we reconsider the introduction of cold-stored platelets in blood bank inventories. F1000Res. 2017;**6**:95. DOI: 10.12688/f1000research.10363.1

[33] Berzuini A, Spreafico M,

[34] Wu X, Darlington DN,

810. DOI: 10.1111/bjh.14999

autologous apheresis platelets.

[37] Humbrecht C, Kientz D,

10.1111/trf.14833

tmrv.2018.04.001

Montgomery RK, Liu B, Keesee JD, Scherer MR, et al. Platelets derived from fresh and cold-stored whole blood participate in clot formation in rats with acute traumatic coagulopathy. British Journal of Haematology. 2017;**179**:802-

[35] Stolla M, Fitzpatrick L, Gettinger I, Bailey SL, Pellham E, Christoffel T, et al. In vivo viability of extended 4°C-stored

Transfusion. 2018;**58**:2407-2413. DOI:

[36] Ng MSY, Tung JP, Fraser JF. Platelet storage lesions: What more do we know now. Transfusion Medicine Reviews. 2018;**32**:144-154. DOI: 10.1016/j.

Gachet C. Platelet transfusion: Current challenges. Transfusion Clinique et Biologique. 2018;**25**:151-164. DOI: 10.1016/j.tracli.2018.06.004

[38] Waters L, Cameron M, Padula MP, Marks DC, Johnson L. Refrigeration, cryopreservation and pathogen

inactivation: An updated perspective on platelet storage conditions. Vox Sanguinis. 2018;**113**:317-328. DOI: 10.1111/vox.12640

[39] Reddoch-Cardenas KM, Sharma U, Salgado CL, Montgomery RK, Cantu C, Cingoz N, et al. An in vitro pilot study of apheresis platelets collected on Trima Accel system and stored in T-PAS+ solution at refrigeration temperature (1-6°C). Transfusion.

**44**

[46] Scott MD, Eaton JW. Antigenic modulation of cells. US Patent Number: 5,908,624. Albany, NY, USA: Assignee Albany Medical College. 1999

[47] Scott MD, Eaton JW. Antigenic modulation of cells. US Patent Number: 8,007,784. Assignee Albany Medical College. 2011

[48] Scott MD, Maurer E. Cold Storage Of Modified Platelets At >0°C. US Patent Number: 7,964,339. Assignee Canadian Blood Services. 2011

[49] Maurer E, Scott MD, Kitamura N. Cold storage of pegylated platelets at about or below 0°C. US Patent Number: 8,067,151. Assignee Canadian Blood Services. 2011

[50] Scott MD, Murad KL, Koumpouras F, Talbot M, Eaton JW. Chemical camouflage of antigenic determinants: Stealth erythrocytes. Proceedings of the National Academy of Sciences of the United States of America. 1997;**94**:7566-7571. DOI: 10.1073/pnas.94.14.7566

[51] Murad KL, Gosselin EJ, Eaton JW, Scott MD. Stealth cells: Prevention of major histocompatibility complex class II-mediated T-cell activation by cell surface modification. Blood. 1999;**94**:2135-2141

[52] Murad KL, Mahany KL, Brugnara C, Kuypers FA, Eaton JW, Scott MD. Structural and functional consequences of antigenic modulation of red blood cells with methoxypoly(ethylene glycol). Blood. 1999;**93**:2121-2127

[53] Scott MD, Bradley AJ, Murad KL. Camouflaged blood cells: Low-technology bioengineering for transfusion medicine? Transfusion Medicine Reviews. 2000;**14**:53-63. DOI: 10.1016/S0887-7963(00) 80115-7

[54] Chen AM, Scott MD. Current and future applications of immunological attenuation via pegylation of cells and tissue. BioDrugs. 2001;**15**:833-847. DOI: 10.2165/00063030-200115120-00005

[55] Bradley AJ, Test ST, Murad KL, Mitsuyoshi J, Scott MD. Interactions of IgM ABO antibodies and complement with methoxy-PEG-modified human RBCs. Transfusion. 2001;**41**:1225-1233. DOI: 10.1046/j.1537-2995.2001.41101225.x

[56] Bradley AJ, Murad KL, Regan KL, Scott MD. Biophysical consequences of linker chemistry and polymer size on stealth erythrocytes: Size does matter. Biochimica et Biophysica Acta. 2002;**1561**:147-158

[57] Bradley AJ, Scott MD. Separation and purification of methoxypoly(ethylene glycol) grafted red blood cells via twophase partitioning. Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences. 2004;**807**:163-168. DOI: 10.1016/j.jchromb.2004.03.054

[58] Chen AM, Scott MD. Comparative analysis of polymer and linker chemistries on the efficacy of immunocamouflage of murine leukocytes. Artificial Cells, Blood Substitutes, and Immobilization Biotechnology. 2006;**34**:305-322. DOI: 10.1080/10731190600683845

[59] Bradley AJ, Scott MD. Immune complex binding by immunocamouflaged [poly(ethylene glycol)-grafted] erythrocytes. American Journal of Hematology. 2007;**82**:970-975. DOI: 10.1002/ajh.20956

## [60] Le Y, Scott MD.

Immunocamouflage: The biophysical basis of immunoprotection by grafted methoxypoly(ethylene glycol) (mPEG). Acta Biomaterialia. 2010;**6**:2631-2641. DOI: 10.1016/j.actbio.2010.01.031

[61] Wang D, Toyofuku WM, Chen AM, Scott MD. Induction of immunotolerance via mPEG grafting to allogeneic leukocytes. Biomaterials. 2011;**32**:9494-9503. DOI: 10.1016/j. biomaterials.2011.08.061

[62] Greco CA, Maurer-Spurej E, Scott MD, Kalab M, Nakane N, Ramirez-Arcos SM. PEGylation prevents bacteria-induced platelet activation and biofilm formation in platelet concentrates. Vox Sanguinis. 2011;**100**:336-339. DOI: 10.1111/j.1423-0410.2010.01419.x

[63] Wang D, Kyluik DL, Murad KL, Toyofuku WM, Scott MD. Polymermediated immunocamouflage of red blood cells: Effects of polymer size on antigenic and immunogenic recognition of allogeneic donor blood cells. Science China. Life Sciences. 2011;**54**:589-598. DOI: 10.1007/ s11427-011-4190-x

[64] Le Y, Li L, Wang D, Scott MD. Immunocamouflage of latex surfaces by grafted methoxypoly(ethylene glycol) (mPEG): Proteomic analysis of plasma protein adsorption. Science China. Life Sciences. 2012;**55**:191-201. DOI: 10.1007/ s11427-012-4290-2

[65] Wang D, Toyofuku WM, Scott MD. The potential utility of methoxypoly(ethylene glycol)-mediated prevention of rhesus blood group antigen RhD recognition in transfusion medicine. Biomaterials. 2012;**33**:3002-3012. DOI: 10.1016/j. biomaterials.2011.12.041

[66] Kyluik-Price DL, Li L, Scott MD. Comparative efficacy of blood cell immunocamouflage by membrane grafting of methoxypoly(ethylene glycol) and polyethyloxazoline. Biomaterials. 2014;**35**:412-422. DOI: 10.1016/j.biomaterials.2013.09.016

[67] Li L, Noumsi GT, Kwok YY, Moulds JM, Scott MD. Inhibition of phagocytic recognition of anti-D opsonized Rh D+ RBC by polymermediated immunocamouflage. American Journal of Hematology. 2015;**90**:1165-1170. DOI: 10.1002/ ajh.24211

[68] Kyluik-Price DL, Scott MD. Effects of methoxypoly (ethylene glycol) mediated immunocamouflage on leukocyte surface marker detection, cell conjugation, activation and alloproliferation. Biomaterials. 2016;**74**:167-177. DOI: 10.1016/j. biomaterials.2015.09.047

[69] Le Y, Toyofuku WM, Scott MD. Immunogenicity of murine mPEGred blood cells and the risk of anti-PEG antibodies in human blood donors. Experimental Hematology. 2017;**47**:36-47.e2. DOI: 10.1016/j. exphem.2016.11.001

[70] Kang N, Toyofuku WM, Yang X, Scott MD. Inhibition of allogeneic cytotoxic T cell (CD8(+)) proliferation via polymer-induced Treg (CD4(+)) cells. Acta Biomaterialia. 2017;**57**:146-155. DOI: 10.1016/j. actbio.2017.04.025

[71] Scott M, Toyofuku W, Yang X, Raj M, Kang N. Immunocamouflaged RBC for alloimmunized patients. In: Koopman-van Gemert A, editor. Transfusion Medicine and Scientific Developments. Croatia: INTECH; 2017. pp. 23-42. DOI: 10.5772/ intechopen.68647

[72] Takahashi T, Hirsh A, Erbe E, Williams RJ. Mechanism of cryoprotection by extracellular polymeric solutes. Biophysical Journal. 1988;**54**:509-518. DOI: 10.1016/ S0006-3495(88)82983-7

[73] Tormanen CD. Cryoprotection of purified rat kidney transamidinase by polyethylene glycol.

**47**

*Cryoprotection of Platelets by Grafted Polymers DOI: http://dx.doi.org/10.5772/intechopen.89272*

> storage requires complementary proteomic approaches. Transfusion.

10.1111/j.1537-2995.2007.01546.x

Platelet storage lesion: A new understanding from a proteomic perspective. Transfusion Medicine Reviews. 2008;**22**:268-279. DOI: 10.1016/j.tmrv.2008.05.004

[81] Thon JN, Schubert P, Devine DV.

[82] Schubert P, Thon JN, Walsh GM, Chen CH, Moore ED, Devine DV, et al. A signaling pathway contributing to platelet storage lesion development: Targeting PI3-kinase-dependent Rap1 activation slows storageinduced platelet deterioration.

Transfusion. 2009;**49**:1944-1955. DOI: 10.1111/j.1537-2995.2009.02224.x

Labrie A, Marziali A, Glatter O. Portable dynamic light scattering instrument and method for the measurement of blood platelet suspensions. Physics in Medicine and Biology. 2006;**51**:3747- 3758. DOI: 10.1088/0031-9155/51/15/010

[83] Maurer-Spurej E, Brown K,

Chipperfield K. Past and future approaches to assess the quality of platelets for transfusion. Transfusion Medicine Reviews. 2007;**21**:295-306. DOI: 10.1016/j.tmrv.2007.05.005

[85] Maurer-Spurej E, Labrie A, Pittendreigh C, Chipperfield K, Smith C, Heddle N, et al. Platelet quality measured with dynamic light scattering correlates with transfusion outcome in hematologic malignancies. Transfusion. 2009;**49**:2276-2284. DOI: 10.1111/j.1537-2995.2009.02302.x

[86] Maurer-Spurej E, Pittendreigh C,

Chipperfield K. Erroneous automated optical platelet counts in 1-hour post-transfusion blood samples. International Journal of Laboratory Hematology. 2010;**32**:e1-e8. DOI: 10.1111/j.1751-553X.2008.01097.x

Yakimec J, De Badyn MH,

[84] Maurer-Spurej E,

2008;**48**:425-435. DOI:

Cryobiology. 1992;**29**:511-518. DOI: 10.1016/0011-2240(92)90054-6

[74] Banker MC, Layne JRJ, Hicks GLJ, Wang TC. Freezing preservation of the mammalian cardiac explant. II. Comparing the protective effect of glycerol and polyethylene glycol. Cryobiology. 1992;**29**:87-94. DOI: 10.1016/0011-2240(92)90008-P

[75] Banker MC, Layne JRJ, Hicks GLJ, Wang T. Freezing preservation of the mammalian heart explant. III. Tissue dehydration and cryoprotection by polyethylene glycol. The Journal of Heart and Lung Transplantation.

[76] Coundouris JA, Grant MH, Engeset J, Petrie JC, Hawksworth GM. Cryopreservation of human adult hepatocytes for use in drug metabolism and toxicity studies. Xenobiotica.

[77] Tsitsanou KE, Oikonomakos NG,

Gregoriou M, Watson KA, et al. Effects of commonly used cryoprotectants on glycogen phosphorylase activity and structure. Protein Science. 1999;**8**:741-

dehydrogenase during freeze-thawing. The AAPS Journal. 2004;**6**:e22. DOI:

[79] Chen YF, Tate MW, Gruner SM.

cryoprotection in thick-walled plastic capillaries by high-pressure cryocooling. Journal of Applied Crystallography. 2009;**42**:525-530. DOI: 10.1107/

[80] Thon JN, Schubert P, Duguay M,

Serrano K, Lin S, Kast J, et al. Comprehensive proteomic analysis of protein changes during platelet

1993;**23**:1399-1409. DOI: 10.3109/00498259309059449

Zographos SE, Skamnaki VT,

749. DOI: 10.1110/ps.8.4.741

[78] Mi Y, Wood G, Thoma L. Cryoprotection mechanisms of polyethylene glycols on lactate

10.1208/aapsj060322

S0021889809011315

Facilitating protein crystal

1992;**11**:619-623

*Cryoprotection of Platelets by Grafted Polymers DOI: http://dx.doi.org/10.5772/intechopen.89272*

Cryobiology. 1992;**29**:511-518. DOI: 10.1016/0011-2240(92)90054-6

*Cryopreservation - Current Advances and Evaluations*

[67] Li L, Noumsi GT, Kwok YY, Moulds JM, Scott MD. Inhibition of phagocytic recognition of anti-D opsonized Rh D+ RBC by polymermediated immunocamouflage. American Journal of Hematology. 2015;**90**:1165-1170. DOI: 10.1002/

[68] Kyluik-Price DL, Scott MD. Effects of methoxypoly (ethylene glycol) mediated immunocamouflage on leukocyte surface marker detection, cell conjugation, activation and alloproliferation. Biomaterials. 2016;**74**:167-177. DOI: 10.1016/j. biomaterials.2015.09.047

[69] Le Y, Toyofuku WM, Scott MD. Immunogenicity of murine mPEGred blood cells and the risk of anti-PEG antibodies in human blood donors. Experimental Hematology. 2017;**47**:36-47.e2. DOI: 10.1016/j.

exphem.2016.11.001

actbio.2017.04.025

intechopen.68647

[72] Takahashi T, Hirsh A,

by polyethylene glycol.

Erbe E, Williams RJ. Mechanism of cryoprotection by extracellular

1988;**54**:509-518. DOI: 10.1016/ S0006-3495(88)82983-7

polymeric solutes. Biophysical Journal.

[73] Tormanen CD. Cryoprotection of purified rat kidney transamidinase

[70] Kang N, Toyofuku WM, Yang X, Scott MD. Inhibition of allogeneic cytotoxic T cell (CD8(+)) proliferation via polymer-induced Treg (CD4(+)) cells. Acta Biomaterialia. 2017;**57**:146-155. DOI: 10.1016/j.

[71] Scott M, Toyofuku W, Yang X, Raj M, Kang N. Immunocamouflaged RBC for alloimmunized patients. In: Koopman-van Gemert A, editor. Transfusion Medicine and Scientific Developments. Croatia: INTECH; 2017. pp. 23-42. DOI: 10.5772/

ajh.24211

Acta Biomaterialia. 2010;**6**:2631-2641. DOI: 10.1016/j.actbio.2010.01.031

[61] Wang D, Toyofuku WM, Chen AM, Scott MD. Induction of immunotolerance via mPEG grafting to allogeneic leukocytes. Biomaterials. 2011;**32**:9494-9503. DOI: 10.1016/j.

biomaterials.2011.08.061

bacteria-induced platelet

s11427-011-4190-x

s11427-012-4290-2

[65] Wang D, Toyofuku WM, Scott MD. The potential utility of methoxypoly(ethylene glycol)-mediated prevention of rhesus blood group antigen RhD recognition in

biomaterials.2011.12.041

transfusion medicine. Biomaterials. 2012;**33**:3002-3012. DOI: 10.1016/j.

[66] Kyluik-Price DL, Li L, Scott MD. Comparative efficacy of blood cell immunocamouflage by membrane grafting of methoxypoly(ethylene glycol) and polyethyloxazoline. Biomaterials. 2014;**35**:412-422. DOI: 10.1016/j.biomaterials.2013.09.016

[62] Greco CA, Maurer-Spurej E, Scott MD, Kalab M, Nakane N,

activation and biofilm formation in platelet concentrates. Vox Sanguinis. 2011;**100**:336-339. DOI: 10.1111/j.1423-0410.2010.01419.x

[63] Wang D, Kyluik DL, Murad KL, Toyofuku WM, Scott MD. Polymermediated immunocamouflage of red blood cells: Effects of polymer size on antigenic and immunogenic recognition of allogeneic donor blood cells. Science China. Life Sciences. 2011;**54**:589-598. DOI: 10.1007/

[64] Le Y, Li L, Wang D, Scott MD. Immunocamouflage of latex surfaces by grafted methoxypoly(ethylene glycol) (mPEG): Proteomic analysis of plasma protein adsorption. Science China. Life Sciences. 2012;**55**:191-201. DOI: 10.1007/

Ramirez-Arcos SM. PEGylation prevents

**46**

[74] Banker MC, Layne JRJ, Hicks GLJ, Wang TC. Freezing preservation of the mammalian cardiac explant. II. Comparing the protective effect of glycerol and polyethylene glycol. Cryobiology. 1992;**29**:87-94. DOI: 10.1016/0011-2240(92)90008-P

[75] Banker MC, Layne JRJ, Hicks GLJ, Wang T. Freezing preservation of the mammalian heart explant. III. Tissue dehydration and cryoprotection by polyethylene glycol. The Journal of Heart and Lung Transplantation. 1992;**11**:619-623

[76] Coundouris JA, Grant MH, Engeset J, Petrie JC, Hawksworth GM. Cryopreservation of human adult hepatocytes for use in drug metabolism and toxicity studies. Xenobiotica. 1993;**23**:1399-1409. DOI: 10.3109/00498259309059449

[77] Tsitsanou KE, Oikonomakos NG, Zographos SE, Skamnaki VT, Gregoriou M, Watson KA, et al. Effects of commonly used cryoprotectants on glycogen phosphorylase activity and structure. Protein Science. 1999;**8**:741- 749. DOI: 10.1110/ps.8.4.741

[78] Mi Y, Wood G, Thoma L. Cryoprotection mechanisms of polyethylene glycols on lactate dehydrogenase during freeze-thawing. The AAPS Journal. 2004;**6**:e22. DOI: 10.1208/aapsj060322

[79] Chen YF, Tate MW, Gruner SM. Facilitating protein crystal cryoprotection in thick-walled plastic capillaries by high-pressure cryocooling. Journal of Applied Crystallography. 2009;**42**:525-530. DOI: 10.1107/ S0021889809011315

[80] Thon JN, Schubert P, Duguay M, Serrano K, Lin S, Kast J, et al. Comprehensive proteomic analysis of protein changes during platelet

storage requires complementary proteomic approaches. Transfusion. 2008;**48**:425-435. DOI: 10.1111/j.1537-2995.2007.01546.x

[81] Thon JN, Schubert P, Devine DV. Platelet storage lesion: A new understanding from a proteomic perspective. Transfusion Medicine Reviews. 2008;**22**:268-279. DOI: 10.1016/j.tmrv.2008.05.004

[82] Schubert P, Thon JN, Walsh GM, Chen CH, Moore ED, Devine DV, et al. A signaling pathway contributing to platelet storage lesion development: Targeting PI3-kinase-dependent Rap1 activation slows storageinduced platelet deterioration. Transfusion. 2009;**49**:1944-1955. DOI: 10.1111/j.1537-2995.2009.02224.x

[83] Maurer-Spurej E, Brown K, Labrie A, Marziali A, Glatter O. Portable dynamic light scattering instrument and method for the measurement of blood platelet suspensions. Physics in Medicine and Biology. 2006;**51**:3747- 3758. DOI: 10.1088/0031-9155/51/15/010

[84] Maurer-Spurej E, Chipperfield K. Past and future approaches to assess the quality of platelets for transfusion. Transfusion Medicine Reviews. 2007;**21**:295-306. DOI: 10.1016/j.tmrv.2007.05.005

[85] Maurer-Spurej E, Labrie A, Pittendreigh C, Chipperfield K, Smith C, Heddle N, et al. Platelet quality measured with dynamic light scattering correlates with transfusion outcome in hematologic malignancies. Transfusion. 2009;**49**:2276-2284. DOI: 10.1111/j.1537-2995.2009.02302.x

[86] Maurer-Spurej E, Pittendreigh C, Yakimec J, De Badyn MH, Chipperfield K. Erroneous automated optical platelet counts in 1-hour post-transfusion blood samples. International Journal of Laboratory Hematology. 2010;**32**:e1-e8. DOI: 10.1111/j.1751-553X.2008.01097.x

[87] Xu Y, Nakane N, Maurer-Spurej E. Novel test for microparticles in platelet-rich plasma and platelet concentrates using dynamic light scattering. Transfusion. 2011;**51**:363-370. DOI: 10.1111/j.1537-2995.2010.02819.x

[88] Labrie A, Marshall A, Bedi H, Maurer-Spurej E. Characterization of platelet concentrates using dynamic light scattering. Transfusion Medicine and Hemotherapy. 2013;**40**:93-100. DOI: 10.1159/000350362

[89] Maurer-Spurej E, Chipperfield K. Could microparticles Be the universal quality indicator for platelet viability and function. Journal of Blood Transfusion. 2016;**2016**:6140239. DOI: 10.1155/2016/6140239

[90] Maurer-Spurej E, Larsen R, Labrie A, Heaton A, Chipperfield K. Microparticle content of platelet concentrates is predicted by donor microparticles and is altered by production methods and stress. Transfusion and Apheresis Science. 2016;**55**:35-43. DOI: 10.1016/j. transci.2016.07.010

[91] Kanzler P, Mahoney A, Leitner G, Witt V, Maurer-Spurej E. Microparticle detection to guide platelet management for the reduction of platelet refractoriness in children - a study proposal. Transfusion and Apheresis Science. 2017;**56**:39-44. DOI: 10.1016/j. transci.2016.12.016

[92] Millar D, Murphy L, Labrie A, Maurer-Spurej E. Routine screening method for microparticles in platelet transfusions. Journal of Visualized Experiments. 2018:e56893. DOI: 10.3791/56893

**49**

**Chapter 3**

**Abstract**

purposes.

**1. Introduction**

protection, formula, additive agents

biomolecular profile of the cells.

*Noha A. Al-Otaibi*

Cryomedia Formula: Cellular

The growing market of cell therapy medicinal products (CTMPs) and biopharmaceuticals demand effective cryopreservation with greater safety, of which the currently available cryoprotective agents [CPAs (e.g., dimethyl sulfoxide, glycerol, trehalose, etc.)] alone are unable to provide. This is due to the need of applying high concentration of CPAs to achieve verification that concomitant oxidative damages. Formulating cocktail of compounds with anti-freezing and antioxidants properties found to be advantageous to overcome the resultant damages. Each cocktail, however, demonstrate overlapping and/or unique protective and modulation effect patterns. The advance technology and research tools (e.g., OMICs) provide a deep insight on how the formulation of cryomedia can influence the cellular pathways and molecular interactions. In fact, this shed the light over the uniqueness of cryomedia formulation and how can they serve various application

**Keywords:** mammalian cells, cryoprotective agents, biological profile, toxicity,

Cryopreservation is one of the most effective techniques that widely used for preserving living cells and organs in research and therapeutic industries [1]. The principle of cryopreservation is to protect cells from the application of super low temperature and ice crystal formation by using media that consist of antifreezing or cryoprotective (CPA) substances such as; glycerol, dimethylsulfoxide (DSMO) or trehalose. The expansion in clinical experiments for medical applications revealed the limitations of utilizing the conventional CPAs which resulting sub-optimal cell quality. This is attributed to the detrimental effects of conventional CPAs and their molecular interactions that compromise cell quality. The new research areas and advanced techniques significantly increase the demand of better cryopreservation

Current trends in cryopreservation are actively focusing on identifying a safe and effective alternative CPAs to substitute or support the conventional agents. In addition, there are various cell types valuable for investigation and medical development and their different biological profile and functional mechanisms required customizing cryopreservation. However, there are limiting number of studies addressing the influence of the cryomedia formulation on the global proteomic and

to maintain the quality and functionality of cells and tissues.

Molecular Perspective

## **Chapter 3**

*Cryopreservation - Current Advances and Evaluations*

[87] Xu Y, Nakane N,

Maurer-Spurej E. Novel test for microparticles in platelet-rich plasma and platelet concentrates using dynamic light scattering. Transfusion. 2011;**51**:363-370. DOI: 10.1111/j.1537-2995.2010.02819.x

[88] Labrie A, Marshall A, Bedi H, Maurer-Spurej E. Characterization of platelet concentrates using dynamic light scattering. Transfusion Medicine and Hemotherapy. 2013;**40**:93-100.

Chipperfield K. Could microparticles Be the universal quality indicator for platelet viability and function. Journal of Blood Transfusion. 2016;**2016**:6140239. DOI: 10.1155/2016/6140239

[90] Maurer-Spurej E, Larsen R, Labrie A, Heaton A, Chipperfield K. Microparticle content of platelet concentrates is predicted by donor microparticles and is altered by production methods and stress. Transfusion and Apheresis Science. 2016;**55**:35-43. DOI: 10.1016/j.

[91] Kanzler P, Mahoney A, Leitner G, Witt V, Maurer-Spurej E. Microparticle detection to guide platelet management

for the reduction of platelet refractoriness in children - a study proposal. Transfusion and Apheresis Science. 2017;**56**:39-44. DOI: 10.1016/j.

[92] Millar D, Murphy L, Labrie A, Maurer-Spurej E. Routine screening method for microparticles in platelet transfusions. Journal of Visualized Experiments. 2018:e56893. DOI:

DOI: 10.1159/000350362

[89] Maurer-Spurej E,

transci.2016.07.010

transci.2016.12.016

10.3791/56893

**48**

## Cryomedia Formula: Cellular Molecular Perspective

*Noha A. Al-Otaibi*

## **Abstract**

The growing market of cell therapy medicinal products (CTMPs) and biopharmaceuticals demand effective cryopreservation with greater safety, of which the currently available cryoprotective agents [CPAs (e.g., dimethyl sulfoxide, glycerol, trehalose, etc.)] alone are unable to provide. This is due to the need of applying high concentration of CPAs to achieve verification that concomitant oxidative damages. Formulating cocktail of compounds with anti-freezing and antioxidants properties found to be advantageous to overcome the resultant damages. Each cocktail, however, demonstrate overlapping and/or unique protective and modulation effect patterns. The advance technology and research tools (e.g., OMICs) provide a deep insight on how the formulation of cryomedia can influence the cellular pathways and molecular interactions. In fact, this shed the light over the uniqueness of cryomedia formulation and how can they serve various application purposes.

**Keywords:** mammalian cells, cryoprotective agents, biological profile, toxicity, protection, formula, additive agents

## **1. Introduction**

Cryopreservation is one of the most effective techniques that widely used for preserving living cells and organs in research and therapeutic industries [1]. The principle of cryopreservation is to protect cells from the application of super low temperature and ice crystal formation by using media that consist of antifreezing or cryoprotective (CPA) substances such as; glycerol, dimethylsulfoxide (DSMO) or trehalose. The expansion in clinical experiments for medical applications revealed the limitations of utilizing the conventional CPAs which resulting sub-optimal cell quality. This is attributed to the detrimental effects of conventional CPAs and their molecular interactions that compromise cell quality. The new research areas and advanced techniques significantly increase the demand of better cryopreservation to maintain the quality and functionality of cells and tissues.

Current trends in cryopreservation are actively focusing on identifying a safe and effective alternative CPAs to substitute or support the conventional agents. In addition, there are various cell types valuable for investigation and medical development and their different biological profile and functional mechanisms required customizing cryopreservation. However, there are limiting number of studies addressing the influence of the cryomedia formulation on the global proteomic and biomolecular profile of the cells.

## **2. Conventional cryomedia compositions and protection mechanisms**

Cryomedia formulation usually encompass of cryoprotective agents (CPAs) and carrier media prepared at or close to cell isotonic concentrations to provide support to cells at low temperature [2]. It also may contain salts, pH buffers, osmotic agents, nutrients, antioxidants or apoptosis inhibitors [2]. There are about 56 CPAs commonly used for different cell cryopreservation [3, 4]. Glycerol and dimethylsulfoxide (DMSO) are the most common CPAs used in cryomedia formula. CPAs are classified based on the permeability through cell membrane into, permeable (pCPA), and impermeable (ipCPA).

pCPAs are generally small, non-ionic molecules that are highly soluble in water, even at low temperatures. They can pass through cellular membranes and equilibrate within the cytoplasm in exchange for intracellular water during dehydration without over dehydrating the cell [5]. They become solid at a lower temperature than water freezing point and subsequently suppress ice crystal formation [6] and mitigate cellular physical damage that could occur in cellular compartments and membranes. Moreover, pCPAs reduce salt-induced stress by dissolving solute and reducing concentrations in the remaining water fraction intracellularly until the cell is cooled to a sufficiently low temperature [6, 7]. pCPA permeability is controlled by their viscosity and the membrane properties of the cell itself [8, 9]. The latter mentioned is variable between different cell types as well as the varying ages of cells [10, 11]. Most of the pCPAs are polyols, such as glycerol and dimethylsulfoxide (DMSO), which are prominence in cryopreservation. Many successful cryopreserving protocols utilized these compounds for their high efficiency in compare to others such as methanol and ethylene glycol [6].

ipCPAs are large molecules usually comprised of long chains of polymers that are unable to permeate through cellular membranes. They are water soluble and thought to increase the osmolarity around cells, which result in cellular dehydration and reduce ice crystal formation intracellularly [12]. The combination of high concentrations of ipCPAs and fast cooling promotes vitrification and stabilizing cellular proteins and membranes [13, 14]. Their protective mechanism is based on preventing ice formation extracellularly as well as intracellular through dehydration [15]. There are several classes of ipCPAs, such as certain forms of sugars, macromolecules, and polymers. Sugars are classified based on their chemical structure into: mono-, di- and poly-saccharides (glucose, trehalose, and raffinose, respectively). A number of these sugars are permeable (e.g., glucose) and others are impermeable (e.g., trehalose). Sugars have garnered unique interest over the last decades. They have been found to protect protein activity and reduce thermal denaturing heat capacity of chemicals [16–21], which leads to protein stabilization. In particular, trehalose has been identified as a universal protein stabilizer and been involved in many freezing and desiccation studies [18–20].

Depending on the freezing mode (slow cooling or vitrification), the concentrations of CPAs are variant in the solution. For instance, slow freezing mode requires less CPA concentration than vitrification. Choosing the optimum concentration of CPA in combination with the cooling rate is crucial for successful cryopreservation [20]. For instance, when preserving human ovarian tissue following slow freezing protocol, DMSO is used with initial concentration of 7.5% and gradually increased to 12.5%. Whereas preserving the same tissue using the vitrification protocol, 20% DMSO is needed [21]. Different tissues and cells, however, demonstrate different responses to the cryopreservation approaches and CPAs. Therefore, the selection of the appropriate protocol and CPA is subject to the cryopreservation empirical success of the desired cells or tissues.

**51**

bovine blastocytes [34].

*Cryomedia Formula: Cellular Molecular Perspective DOI: http://dx.doi.org/10.5772/intechopen.91382*

**protection action**

**3. Quality assessment methods of cryopreserved cells and CPA** 

changes that impact cryopreserved cells' function and morphology.

probes that influencing the accuracy of the measurement [25].

**4. Cryoprotectant toxicity and detrimental effects**

An accurate assessment of cryopreserved cells or tissues considering the viability and functionality is paramount to determine the quality and reliability of the cryopreservation protocol and solution. In the past, classical parameters, such as survival rate or motility, were the only quality measurements [22, 23]. With the evolution in technologies and developed assays, scientists can obtain more information surrounding the level of stress that heralds cellular death cascades and dynamic

Nowadays, there are a wide range of viability assays available; however, selecting

The emergence of developed technologies, such as genomics, transcriptomics,

Introducing CPAs in high concentration (molars) is accompanied with nonspecific adverse effects such as osmotic stress and cell dehydration [29] that also could induce the oxidative stress [30]. This can cause severe cell damage; for instance, increasing the concentration of DMSO, glycerol, and 1,2-propanediol is linked with the production of non-enzymatic formaldehyde [31], a cytotoxic compound that contributes to cell death [32]. The long exposure duration of cells to high concentration of CPA also harm cell development, as reported when exposing bovine blastocytes to a high concentration of ethylene glycol over 10 min [33]. Likewise, introducing a high concentration of propanediol to mouse zygotes was found to have a similar damaging effect on cell development to that observed in

The high concentration of CPA accumulated intracellularly has a detrimental effect on cells. In cryopreserved human mesenchymal stem cells (hMSC), it has a significant effect on cellular viability, filamentous actin distribution, intracellular pH, and mitochondria aggregation [35]. It has also been found to cause abnormal spindles and morphology in human oocytes, which can potentially influence their viability post-cryopreservation [36]. Similarly, CPA causes a serious alteration in mammalian sperm viability, physiological properties, protein phosphorylation patterns [37], and can lethally damage enzymatic activity and DNA [38]. However, osmotic stress factors and associated cell shock cannot be decoupled since they interact with each other, though the resultant effects can be reversed or limited to

proteomics, and metabolomics (collectively termed OMICs), has provided a comprehensive profile of cryopreserved cells, including their stressed and compromised biological pathways, which may help designing protocols or solutions in order to modulate the damaged pathways. So far, the majority of OMICs applications in cryopreservation are limited to reproduction medicine and plants [26, 27], such as in human sperm characterization post-thaw [28]. The deep analysis OMICs stresses the importance of adopting such analytical approach in researches aiming at advancing cryopreservation and biobanking for better CTMPs outcome [25].

the appropriate assay mainly depends on cell types to avoid inaccurate measurement. For instance, the measurement of LDH leakage in media can be used for membrane integrity assessment because of its reliability and easy performance. It is an applicable measurement in single cells as well as tissues and organs [24]. Conversely, using fluorescent probes for viability measurement is suitable for many cells excluding hepatocytes, because of their detoxification activity with respect to

*Cryopreservation - Current Advances and Evaluations*

(pCPA), and impermeable (ipCPA).

and ethylene glycol [6].

many freezing and desiccation studies [18–20].

success of the desired cells or tissues.

**2. Conventional cryomedia compositions and protection mechanisms**

Cryomedia formulation usually encompass of cryoprotective agents (CPAs) and carrier media prepared at or close to cell isotonic concentrations to provide support to cells at low temperature [2]. It also may contain salts, pH buffers, osmotic agents, nutrients, antioxidants or apoptosis inhibitors [2]. There are about 56 CPAs commonly used for different cell cryopreservation [3, 4]. Glycerol and dimethylsulfoxide (DMSO) are the most common CPAs used in cryomedia formula. CPAs are classified based on the permeability through cell membrane into, permeable

pCPAs are generally small, non-ionic molecules that are highly soluble in water, even at low temperatures. They can pass through cellular membranes and equilibrate within the cytoplasm in exchange for intracellular water during dehydration without over dehydrating the cell [5]. They become solid at a lower temperature than water freezing point and subsequently suppress ice crystal formation [6] and mitigate cellular physical damage that could occur in cellular compartments and membranes. Moreover, pCPAs reduce salt-induced stress by dissolving solute and reducing concentrations in the remaining water fraction intracellularly until the cell is cooled to a sufficiently low temperature [6, 7]. pCPA permeability is controlled by their viscosity and the membrane properties of the cell itself [8, 9]. The latter mentioned is variable between different cell types as well as the varying ages of cells [10, 11]. Most of the pCPAs are polyols, such as glycerol and dimethylsulfoxide (DMSO), which are prominence in cryopreservation. Many successful cryopreserving protocols utilized these compounds for their high efficiency in compare to others such as methanol

ipCPAs are large molecules usually comprised of long chains of polymers that are unable to permeate through cellular membranes. They are water soluble and thought to increase the osmolarity around cells, which result in cellular dehydration and reduce ice crystal formation intracellularly [12]. The combination of high concentrations of ipCPAs and fast cooling promotes vitrification and stabilizing cellular proteins and membranes [13, 14]. Their protective mechanism is based on preventing ice formation extracellularly as well as intracellular through dehydration [15]. There are several classes of ipCPAs, such as certain forms of sugars, macromolecules, and polymers. Sugars are classified based on their chemical structure into: mono-, di- and poly-saccharides (glucose, trehalose, and raffinose, respectively). A number of these sugars are permeable (e.g., glucose) and others are impermeable (e.g., trehalose). Sugars have garnered unique interest over the last decades. They have been found to protect protein activity and reduce thermal denaturing heat capacity of chemicals [16–21], which leads to protein stabilization. In particular, trehalose has been identified as a universal protein stabilizer and been involved in

Depending on the freezing mode (slow cooling or vitrification), the concentrations of CPAs are variant in the solution. For instance, slow freezing mode requires less CPA concentration than vitrification. Choosing the optimum concentration of CPA in combination with the cooling rate is crucial for successful cryopreservation [20]. For instance, when preserving human ovarian tissue following slow freezing protocol, DMSO is used with initial concentration of 7.5% and gradually increased to 12.5%. Whereas preserving the same tissue using the vitrification protocol, 20% DMSO is needed [21]. Different tissues and cells, however, demonstrate different responses to the cryopreservation approaches and CPAs. Therefore, the selection of the appropriate protocol and CPA is subject to the cryopreservation empirical

**50**

## **3. Quality assessment methods of cryopreserved cells and CPA protection action**

An accurate assessment of cryopreserved cells or tissues considering the viability and functionality is paramount to determine the quality and reliability of the cryopreservation protocol and solution. In the past, classical parameters, such as survival rate or motility, were the only quality measurements [22, 23]. With the evolution in technologies and developed assays, scientists can obtain more information surrounding the level of stress that heralds cellular death cascades and dynamic changes that impact cryopreserved cells' function and morphology.

Nowadays, there are a wide range of viability assays available; however, selecting the appropriate assay mainly depends on cell types to avoid inaccurate measurement. For instance, the measurement of LDH leakage in media can be used for membrane integrity assessment because of its reliability and easy performance. It is an applicable measurement in single cells as well as tissues and organs [24]. Conversely, using fluorescent probes for viability measurement is suitable for many cells excluding hepatocytes, because of their detoxification activity with respect to probes that influencing the accuracy of the measurement [25].

The emergence of developed technologies, such as genomics, transcriptomics, proteomics, and metabolomics (collectively termed OMICs), has provided a comprehensive profile of cryopreserved cells, including their stressed and compromised biological pathways, which may help designing protocols or solutions in order to modulate the damaged pathways. So far, the majority of OMICs applications in cryopreservation are limited to reproduction medicine and plants [26, 27], such as in human sperm characterization post-thaw [28]. The deep analysis OMICs stresses the importance of adopting such analytical approach in researches aiming at advancing cryopreservation and biobanking for better CTMPs outcome [25].

## **4. Cryoprotectant toxicity and detrimental effects**

Introducing CPAs in high concentration (molars) is accompanied with nonspecific adverse effects such as osmotic stress and cell dehydration [29] that also could induce the oxidative stress [30]. This can cause severe cell damage; for instance, increasing the concentration of DMSO, glycerol, and 1,2-propanediol is linked with the production of non-enzymatic formaldehyde [31], a cytotoxic compound that contributes to cell death [32]. The long exposure duration of cells to high concentration of CPA also harm cell development, as reported when exposing bovine blastocytes to a high concentration of ethylene glycol over 10 min [33]. Likewise, introducing a high concentration of propanediol to mouse zygotes was found to have a similar damaging effect on cell development to that observed in bovine blastocytes [34].

The high concentration of CPA accumulated intracellularly has a detrimental effect on cells. In cryopreserved human mesenchymal stem cells (hMSC), it has a significant effect on cellular viability, filamentous actin distribution, intracellular pH, and mitochondria aggregation [35]. It has also been found to cause abnormal spindles and morphology in human oocytes, which can potentially influence their viability post-cryopreservation [36]. Similarly, CPA causes a serious alteration in mammalian sperm viability, physiological properties, protein phosphorylation patterns [37], and can lethally damage enzymatic activity and DNA [38]. However, osmotic stress factors and associated cell shock cannot be decoupled since they interact with each other, though the resultant effects can be reversed or limited to

a certain extent by minimizing exposure time, accelerating freezing and thawing speeds, and gradually diluting CPAs in cells [39], which can increase post-thaw cell viability. These types of reported damages are considered non-specific since it is not limited to specific CPA identity. However, the molecular interaction of CPAs is more closely linked to the permeable CPAs, as they are able to interact with the cell compartments and biomolecules [30].

CPA toxicity effect can be either reversible (e.g., osmotic shock and cellular shrinkage) [40, 41] or irreversible. Notably, cryopreservation protocols involving short exposure times to CPAs can reverse the induced damages. Nevertheless, irreversible damage is common in cells lacking self-renewal or repair mechanisms, such as RBCs [42] and embryonic stem cells [43, 44].

Oxidative stress occurs during cryopreservation, mainly when adding CPAs to cells [45]. The increased oxidative stress results in more ROS production [46], which leads to a disequilibrium between the generated ROS and the cellular antioxidant capacity within the redox pathway. A decrease in cellular-reduced glutathione (GSH) content was observed during the freezing step of sperm [47], indicating that oxidative damage occurs during the initial steps of cryopreservation. Consequently, increased ROS production results in lipid peroxidation [48], DNA instability [49], protein oxidation [50], overall dysfunctional cells, and low survival rates [47, 49]. Oxidative stress has been observed when applying glycerol [51], DMSO [52], and trehalose [50] to cells.

## **4.1 Other biochemical effects**

Cells naturally have a dynamic and complex system involving active biomolecules that respond distinctly to all forms of environmental stressors, including CPA media and temperature alterations. The cells' response to stressors involves complex biomolecular events influencing their fate. Measuring the survival rate of thawed cells is a classical parameter that is not precise when determining the efficacy of cryopreservation. This is because during the recovery period, a decrease in cellular viability occurs in different cell types [53]. This is attributed to the activation of apoptosis machinery post-thaw [54]. Xu et al. [53] reported that exposing cells to DMSO and freezing conditions activate apoptosis through extrinsic and intrinsic pathways, including caspase-8, caspase-9, and p53. Some CPAs have different mechanisms, yet they lead to the same lethal results. Propylene glycol (ProH), for instance, reduced cell viability via increasing intracellular calcium to a cytotoxic level [55].

Furthermore, the cryopreservation affects cells' biomarkers [56]. It alters the proteome profile of cells, which in some cases can bring about changes in cellular metabolism, function, and structure [57]. In previous work, there is often no clear demarcation between the effect of CPAs and the cryopreservation protocol itself. However, the exact effect of CPAs can be investigated in an experiment if cell viability and functionality are analyzed before freezing.

## **5. Modulating CPA damages via additive agents**

Considering the aforementioned limitations in cryomedia formula, many active studies investigated the efficacy of additive agents to improve the cryomedia and modulate the resultant damages in cryopreserved cells (**Table 1**). Additive agents have variable effects on different cells. This was evidently observed in number of cases such as; quercetin, glutathione, and ascorbic acid [58]. On other hand, some other demonstrated similar efficient antioxidant protection effect on several cells

**53**

viability rate.

**Table 1.**

Anti-apoptotic drugs

*Cryomedia Formula: Cellular Molecular Perspective DOI: http://dx.doi.org/10.5772/intechopen.91382*

Salidroside [62]

anti-freezing proteins [63] Sericin [64]

Sphingosine-1 and Z-VAD-FMK [67]

*Cryomedia additive agents and their effects on cryopreserved cells.*

Enzymes Catalase [65] 40 μl/ml Mice

15 μM 200 μM

0.8 mg/ml

Vitamins Vit E [66] 100–200 μmol Human sperm Increase motility

5%

Antioxidants Resveratrol [61]

Proteins Type III

**Additive agents**

(e.g., curcumin) [58]. Notably, many protective factors share their antioxidant protection effects at different concentrations (e.g., hyaluronan and glutamine [59, 60]) that commonly include reducing oxidative stress on lipid and proteins and improve

**Example Concentration Cell types Molecular and biological** 

Human carcinoma cells Human sperm

spermatogonia stem cells

10 μM Ovarian sheep Preserve primordial

Human sperms Red blood cells

**effects**

thawing

Reduce hemolysis, lactate dehydrogenase activity and protect protein and lipid from oxidation damage

Increase cells recovery post-thawing Increase cells motility Decreased DNA fragmentation

Reduce apoptosis and ROS

follicular density, with normal morphology and improved proliferation

production Increase viability

Decrease DNA fragmentation through activating AMP-activated protein kinase (AMPK) Increase glutathione reductase (GR) activity and cells stability post

In our published studies, the discovery of the protection potent of salidroside and nigerose was exceptional on nucleated as well as anucleated hematopoietic cells [RBCs and human leukemia cells (HL-60)] in various cryomedia formulae and freezing modes. The efficacy of these compounds was evidenced at low concentrations (200–300 μM) of salidroside and nigerose, respectively. The effect of the additive compounds was determined by analyzing both the biomolecular and proteomic profiles of the survival cells [58]. First, we examined the effect of salidroside in standard cryo-solutions (glycerol and trehalose), which are commonly used for the RBCs biopreservation, using RBCs [62]. When comparing the survival cells rate, RBCs cryopreserved in solutions contained salidroside showed higher survival rate in compare to those cryopreserved in standard cryomedia alone. On biomolecular level, salidroside improved the intracellular activity of glutathione reductase (GR), the active enzyme in the redox pathway. In addition, it reduced the level of stress resultant from freeze-thaw process, as it was measured by intracellular lactate dehydrogenase (LDH) activity [68]. Moreover, it protected RBC proteins against oxidative damages [62]. Further investigation on human leukemia cells (HL-60) using salidroside in 2% DMSO and fetal bovine serum cryosolution demonstrated similar protection effects to what have been seen in RBCs [62, 68]. Additionally, it protected lipid against oxidative stress. In the same study, we used nigerose for comparison, which showed similar protection effect on the biomolecular profile of the cells.


## *Cryomedia Formula: Cellular Molecular Perspective DOI: http://dx.doi.org/10.5772/intechopen.91382*

## **Table 1.**

*Cryopreservation - Current Advances and Evaluations*

compartments and biomolecules [30].

trehalose [50] to cells.

**4.1 Other biochemical effects**

such as RBCs [42] and embryonic stem cells [43, 44].

a certain extent by minimizing exposure time, accelerating freezing and thawing speeds, and gradually diluting CPAs in cells [39], which can increase post-thaw cell viability. These types of reported damages are considered non-specific since it is not limited to specific CPA identity. However, the molecular interaction of CPAs is more closely linked to the permeable CPAs, as they are able to interact with the cell

CPA toxicity effect can be either reversible (e.g., osmotic shock and cellular shrinkage) [40, 41] or irreversible. Notably, cryopreservation protocols involving short exposure times to CPAs can reverse the induced damages. Nevertheless, irreversible damage is common in cells lacking self-renewal or repair mechanisms,

Oxidative stress occurs during cryopreservation, mainly when adding CPAs to cells [45]. The increased oxidative stress results in more ROS production [46], which leads to a disequilibrium between the generated ROS and the cellular antioxidant capacity within the redox pathway. A decrease in cellular-reduced glutathione (GSH) content was observed during the freezing step of sperm [47], indicating that oxidative damage occurs during the initial steps of cryopreservation. Consequently, increased ROS production results in lipid peroxidation [48], DNA instability [49], protein oxidation [50], overall dysfunctional cells, and low survival rates [47, 49]. Oxidative stress has been observed when applying glycerol [51], DMSO [52], and

Cells naturally have a dynamic and complex system involving active biomolecules that respond distinctly to all forms of environmental stressors, including CPA media and temperature alterations. The cells' response to stressors involves complex biomolecular events influencing their fate. Measuring the survival rate of thawed cells is a classical parameter that is not precise when determining the efficacy of cryopreservation. This is because during the recovery period, a decrease in cellular viability occurs in different cell types [53]. This is attributed to the activation of apoptosis machinery post-thaw [54]. Xu et al. [53] reported that exposing cells to DMSO and freezing conditions activate apoptosis through extrinsic and intrinsic pathways, including caspase-8, caspase-9, and p53. Some CPAs have different mechanisms, yet they lead to the same lethal results. Propylene glycol (ProH), for instance, reduced cell viability via increasing intracellular calcium to a cytotoxic

Furthermore, the cryopreservation affects cells' biomarkers [56]. It alters the proteome profile of cells, which in some cases can bring about changes in cellular metabolism, function, and structure [57]. In previous work, there is often no clear demarcation between the effect of CPAs and the cryopreservation protocol itself. However, the exact effect of CPAs can be investigated in an experiment if cell

Considering the aforementioned limitations in cryomedia formula, many active studies investigated the efficacy of additive agents to improve the cryomedia and modulate the resultant damages in cryopreserved cells (**Table 1**). Additive agents have variable effects on different cells. This was evidently observed in number of cases such as; quercetin, glutathione, and ascorbic acid [58]. On other hand, some other demonstrated similar efficient antioxidant protection effect on several cells

viability and functionality are analyzed before freezing.

**5. Modulating CPA damages via additive agents**

**52**

level [55].

*Cryomedia additive agents and their effects on cryopreserved cells.*

(e.g., curcumin) [58]. Notably, many protective factors share their antioxidant protection effects at different concentrations (e.g., hyaluronan and glutamine [59, 60]) that commonly include reducing oxidative stress on lipid and proteins and improve viability rate.

In our published studies, the discovery of the protection potent of salidroside and nigerose was exceptional on nucleated as well as anucleated hematopoietic cells [RBCs and human leukemia cells (HL-60)] in various cryomedia formulae and freezing modes. The efficacy of these compounds was evidenced at low concentrations (200–300 μM) of salidroside and nigerose, respectively. The effect of the additive compounds was determined by analyzing both the biomolecular and proteomic profiles of the survival cells [58]. First, we examined the effect of salidroside in standard cryo-solutions (glycerol and trehalose), which are commonly used for the RBCs biopreservation, using RBCs [62]. When comparing the survival cells rate, RBCs cryopreserved in solutions contained salidroside showed higher survival rate in compare to those cryopreserved in standard cryomedia alone. On biomolecular level, salidroside improved the intracellular activity of glutathione reductase (GR), the active enzyme in the redox pathway. In addition, it reduced the level of stress resultant from freeze-thaw process, as it was measured by intracellular lactate dehydrogenase (LDH) activity [68]. Moreover, it protected RBC proteins against oxidative damages [62]. Further investigation on human leukemia cells (HL-60) using salidroside in 2% DMSO and fetal bovine serum cryosolution demonstrated similar protection effects to what have been seen in RBCs [62, 68]. Additionally, it protected lipid against oxidative stress. In the same study, we used nigerose for comparison, which showed similar protection effect on the biomolecular profile of the cells.

On top of the biological profile of cryopreserved cells, proteomic analysis revealed the specific and unique modulation effect of additive agents on compromised biological pathways [68]. Each compound was observed to have a demonstrably unique effect on the proteome pattern of cryopreserved HL-60 cells. Nigerose was strongly engaged with cell maintenance, energetic, and metabolic pathways, whereas salidroside influenced proteins associated with DNA binding and nuclear activities. Both overlapped with regards to influencing proteins associated with redox pathways. Moreover, the damaging effects of classical cryomedia were modulated by the reformulated media comprising the novel protective agents. The protective mechanisms of the compounds on the proteomic level were strongly compatible with the biochemical analysis of the cells cryosurvival rate and their resistance to stressors [68]. This has shed the light over the potency of specific effectiveness of additive agents in the cryosolution and their specific applications for preserving different cells and tissues for pharmaceutical and clinical applications.

## **6. Conclusion**

Understanding of the protective mechanisms of cryomedia ingredients along with identifying powerful protective compounds to enhance cryomedia performance is highly demanded. Due to the wide range of preserved cells and tissues, designing the appropriate cryosolution with suitable protocol is beneficial. In fact, these are particularly important for CTMP industries and end-users at clinics, such as those with cancer and diabetes or requiring blood transfusion, organ transplantation, and infertility treatments.

## **Author details**

Noha A. Al-Otaibi Life Science and Environment Research Institutes, King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia

\*Address all correspondence to: naalotaibi@kacst.edu.sa

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**55**

*Cryomedia Formula: Cellular Molecular Perspective DOI: http://dx.doi.org/10.5772/intechopen.91382*

> characterization of its light-driven Cl− pump activity. Biophysical Journal. 2007;**92**(7):2559-2569. Available at: http://linkinghub.elsevier.com/retrieve/

> [10] Valdez DM, Miyamoto A, Hara T, Seki S, Kasai M, Edashige K. Waterand cryoprotectant-permeability of mature and immature oocytes in the medaka (Oryzias latipes). Cryobiology.

[11] Pedro PB, Yokoyama E, Zhu SE, Yoshida N, Valdez DM, Tanaka M, et al. Permeability of mouse oocytes and embryos at various developmental

stages to five cryoprotectants. The Journal of Reproduction and Development. 2005;**51**(2):235-246

[12] Mohanty JG, Nagababu E. Rifkind JM. Red blood cell oxidative stress impairs oxygen delivery and induces red blood cell aging. Frontiers in Physiology. 2014;**5**:84. Available at: http://www.pubmedcentral.nih.gov/ articlerender.fcgi?artid=3937982&tool= pmcentrez&rendertype=abstract

[13] Crowe JH, Crowe LM, Wolkers WF,

[14] Jain NK, Roy I. Effect of trehalose on protein structure. Protein Science.

[15] Acker JP. The use of intracellular protectants in cell biopreservation. In: Baust JG, Baust JM, editors. Advances in Biopreservation. New york, Taylors and

[16] Colaço C, Sen S, Thangavelu M, Pinder S, Roser B. Extraordinary stability of enzymes dried in trehalose:

Oliver AE, Ma X, Auh J-H, et al. Stabilization of dry mammalian cells: Lessons from nature. Integrative and Comparative Biology. 2005;**45**(5):810- 820. Available at: http://www.ncbi.nlm.

nih.gov/pubmed/21676832

Francis; 2007. pp. 291-313

2009;**18**:24-36

pii/S0006349507710603

2005;**50**(1):93-102

**References**

[1] Taylor MJ, Weegman BP,

Hemotherapy. 2019;**46**:197-215

Cryonics. 2007;**3**:28

Baicu SC, Giwa SE. New approaches to cryopreservation of cells, tissues, and organs. Transfusion Medicine and

[2] Wowk B. How cryoprotectants work.

[3] Karow AM. Cryoprotectants—A new class of drugs. The Journal of Pharmacy and Pharmacology. 1969;**21**(4):209-223

[4] Hubálek Z. Protectants used in the cryopreservation of microorganisms. Cryobiology. 2003;**46**(3):205-229

[5] Lovelock JE. The mechanism of the protective action of glycerol against haemolysis by freezing and thawing. Biochimica et Biophysica Acta.

1953;**11**:28-36. Available at: http://www. sciencedirect.com/science/article/

[6] Swain JE, Smith GD. Cryoprotectants. In: Chain R-C, Quinn P, editors. Fertility Cryopreservation. Cambridge: University of Cambridge Press; 2017. pp. 24-33

[8] Karlsson JOM, Younis AI, Chan AWS, Gould KG, Eroglu A. Permeability of the rhesus monkey oocyte membrane to water and common cryoprotectants.

Molecular Reproduction and Development. 2009;**76**(4):321-333

[9] Seki A, Miyauchi S, Hayashi S, Kikukawa T, Kubo M, Demura M, et al. Heterologous expression of Pharaonis Halorhodopsin in Xenopus laevis oocytes and electrophysiological

pii/0006300253900055

[7] Thanner M, Nagel E. A comprehensive assessment of ATMP. Difficulties and approaches. Bundesgesundheitsblatt - Gesundheitsforschung - Gesundheitsschutz. 2011;**54**(7):843-848. Available at: http://www.ncbi.nlm.nih.gov/

pubmed/21698538

*Cryomedia Formula: Cellular Molecular Perspective DOI: http://dx.doi.org/10.5772/intechopen.91382*

## **References**

*Cryopreservation - Current Advances and Evaluations*

On top of the biological profile of cryopreserved cells, proteomic analysis revealed the specific and unique modulation effect of additive agents on compromised biological pathways [68]. Each compound was observed to have a demonstrably unique effect on the proteome pattern of cryopreserved HL-60 cells. Nigerose was strongly engaged with cell maintenance, energetic, and metabolic pathways, whereas salidroside influenced proteins associated with DNA binding and nuclear activities. Both overlapped with regards to influencing proteins associated with redox pathways. Moreover, the damaging effects of classical cryomedia were modulated by the reformulated media comprising the novel protective agents. The protective mechanisms of the compounds on the proteomic level were strongly compatible with the biochemical analysis of the cells cryosurvival rate and their resistance to stressors [68]. This has shed the light over the potency of specific effectiveness of additive agents in the cryosolution and their specific applications for preserving

different cells and tissues for pharmaceutical and clinical applications.

Understanding of the protective mechanisms of cryomedia ingredients along with identifying powerful protective compounds to enhance cryomedia performance is highly demanded. Due to the wide range of preserved cells and tissues, designing the appropriate cryosolution with suitable protocol is beneficial. In fact, these are particularly important for CTMP industries and end-users at clinics, such as those with cancer and diabetes or requiring blood transfusion, organ transplan-

**54**

**Author details**

**6. Conclusion**

Noha A. Al-Otaibi

and Technology, Riyadh, Saudi Arabia

provided the original work is properly cited.

tation, and infertility treatments.

\*Address all correspondence to: naalotaibi@kacst.edu.sa

Life Science and Environment Research Institutes, King Abdulaziz City for Science

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

[1] Taylor MJ, Weegman BP, Baicu SC, Giwa SE. New approaches to cryopreservation of cells, tissues, and organs. Transfusion Medicine and Hemotherapy. 2019;**46**:197-215

[2] Wowk B. How cryoprotectants work. Cryonics. 2007;**3**:28

[3] Karow AM. Cryoprotectants—A new class of drugs. The Journal of Pharmacy and Pharmacology. 1969;**21**(4):209-223

[4] Hubálek Z. Protectants used in the cryopreservation of microorganisms. Cryobiology. 2003;**46**(3):205-229

[5] Lovelock JE. The mechanism of the protective action of glycerol against haemolysis by freezing and thawing. Biochimica et Biophysica Acta. 1953;**11**:28-36. Available at: http://www. sciencedirect.com/science/article/ pii/0006300253900055

[6] Swain JE, Smith GD. Cryoprotectants. In: Chain R-C, Quinn P, editors. Fertility Cryopreservation. Cambridge: University of Cambridge Press; 2017. pp. 24-33

[7] Thanner M, Nagel E. A comprehensive assessment of ATMP. Difficulties and approaches. Bundesgesundheitsblatt - Gesundheitsforschung - Gesundheitsschutz. 2011;**54**(7):843-848. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/21698538

[8] Karlsson JOM, Younis AI, Chan AWS, Gould KG, Eroglu A. Permeability of the rhesus monkey oocyte membrane to water and common cryoprotectants. Molecular Reproduction and Development. 2009;**76**(4):321-333

[9] Seki A, Miyauchi S, Hayashi S, Kikukawa T, Kubo M, Demura M, et al. Heterologous expression of Pharaonis Halorhodopsin in Xenopus laevis oocytes and electrophysiological

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[10] Valdez DM, Miyamoto A, Hara T, Seki S, Kasai M, Edashige K. Waterand cryoprotectant-permeability of mature and immature oocytes in the medaka (Oryzias latipes). Cryobiology. 2005;**50**(1):93-102

[11] Pedro PB, Yokoyama E, Zhu SE, Yoshida N, Valdez DM, Tanaka M, et al. Permeability of mouse oocytes and embryos at various developmental stages to five cryoprotectants. The Journal of Reproduction and Development. 2005;**51**(2):235-246

[12] Mohanty JG, Nagababu E. Rifkind JM. Red blood cell oxidative stress impairs oxygen delivery and induces red blood cell aging. Frontiers in Physiology. 2014;**5**:84. Available at: http://www.pubmedcentral.nih.gov/ articlerender.fcgi?artid=3937982&tool= pmcentrez&rendertype=abstract

[13] Crowe JH, Crowe LM, Wolkers WF, Oliver AE, Ma X, Auh J-H, et al. Stabilization of dry mammalian cells: Lessons from nature. Integrative and Comparative Biology. 2005;**45**(5):810- 820. Available at: http://www.ncbi.nlm. nih.gov/pubmed/21676832

[14] Jain NK, Roy I. Effect of trehalose on protein structure. Protein Science. 2009;**18**:24-36

[15] Acker JP. The use of intracellular protectants in cell biopreservation. In: Baust JG, Baust JM, editors. Advances in Biopreservation. New york, Taylors and Francis; 2007. pp. 291-313

[16] Colaço C, Sen S, Thangavelu M, Pinder S, Roser B. Extraordinary stability of enzymes dried in trehalose: Simplified molecular biology. Bio/ Technology. 1992;**10**(9):1007-1011

[17] Xie G, Timasheff SN. The Thermodynamic Mechanism of Protein Stabilization by Trehalose. Biophysical Chemistry. Elsevier; 1997. pp. 25-43

[18] Lynch AL, Chen R, NKH S. pH-responsive polymers for trehalose loading and desiccation protection of human red blood cells. Biomaterials. 2011;**32**(19):4443-4449. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/21421265

[19] Sampedro JG, Guerra G, Pardo J-P, Uribe S. Trehalose-mediated protection of the plasma membrane H+-ATPase from Kluyveromyces lactis during freezedrying and rehydration. Cryobiology. 1998;**37**(2):131-138. Available at: http:// www.sciencedirect.com/science/article/ pii/S0011224098921095

[20] Radaelli MRM, Almodin CG, Minguetti-Câmara VC, Cerialli PMA, Nassif AE, Gonçalves AJ. A comparison between a new vitrification protocol and the slow freezing method in the cryopreservation of prepubertal testicular tissue. JBRA Assisted Reproduction. 2017;**21**(3):188-195

[21] Lee S, Ryu K-J, Kim B, Kang D, Kim YY, Kim T. Comparison between slow freezing and Vitrification for human ovarian tissue cryopreservation and xenotransplantation. International Journal of Molecular Sciences. 2019;**20**(13):3346

[22] Deller RC, Vatish M, Mitchell DA, Gibson MI. Glycerol-free cryopreservation of red blood cells enabled by ice-recrystallization-inhibiting polymers. ACS Biomaterials Science & Engineering. 2015;**1**(9):789-794. Available at: http://pubs.acs.org/ doi/10.1021/acsbiomaterials.5b00162

[23] Clarke DM, Yadock DJ, Nicoud IB, Mathew AJ, Heimfeld S. Improved

post-thaw recovery of peripheral blood stem/progenitor cells using a novel intracellular-like cryopreservation solution. Cytotherapy. 2009;**11**(4):472-479

[24] Drent M, NAM C, Henderson RF, EFM W, Van Dieijen-Visser M. Usefulness of lactate dehydrogenase and its isoenzymes as indicators of lung damage or inflammation. European Respiratory Journal. 1996;**9**:1736-1742

[25] Van BRG. Viability and functional assays used to assess preservation efficacy. In: Baust JG, Baust JM, editors. Advances in Biopreservation. Boca Raton: Taylor & Francis; 2007. pp. 123-141

[26] Egea R, Escrivá M, Puchalt N, Varghese A. OMICS: Current and future perspectives in reproductive medicine and technology. Journal of Human Reproductive Sciences. 2014;**7**(2):73. Available at: http://www.jhrsonline.org/ text.asp?2014/7/2/73/138857

[27] Volk GM. Application of functional genomics and proteomics to plant cryopreservation. Current Genomics. 2010;**11**(1):24-29. Available at: http:// www.pubmedcentral.nih.gov/ articlerender.fcgi?artid=2851113&tool= pmcentrez&rendertype=abstract

[28] Wang S, Wang W, Xu Y, Tang M, Fang J, Sun H, et al. Proteomic characteristics of human sperm cryopreservation. Proteomics. 2014;**14**(2-3):298-310

[29] Arakawa T, Carpenter JF, Kita YA, Crowe JH. The basis for toxicity of certain cryoprotectants: A hypothesis. Cryobiology. 1990;**27**(4):401-415

[30] Elliott GD, Wang S, Fuller BJ. Cryoprotectants: A review of the actions and applications of cryoprotective solutes that modulate cell recovery from

**57**

1148-1154

*Cryomedia Formula: Cellular Molecular Perspective DOI: http://dx.doi.org/10.5772/intechopen.91382*

> 2007;**8**(4):209-218. Available at: http://www.ncbi.nlm.nih.gov/

concentration. Transfusion.

[39] Meryman HT, Hornblower M. A method for freezing and washing red blood cells using a high glycerol

[40] Ogura T, Shuba LM, McDonald TF. Action potentials, ionic currents and cell water in Guinea pig ventricular preparations exposed to dimethyl sulfoxide. Journal of Pharmacology and Experimental Therapeutics. 1995;**273**:1273-1286. Available at: http://www.ncbi.nlm.nih.gov/

[41] Pribor DB, Nara A. The effect of salt or various cryoprotective agents on frog sciatic nerves. Cryobiology.

Groenen-Döpp YAM, Roerdinkholder-Stoelwinder B, De Pauw B, Bosman GJCGM. Erythrocyte vesiculation: A self-protective mechanism? British Journal of Haematology.

[42] Willekens FLA, Werre JM,

pubmed/18645598

1972;**12**(3):145-156

pubmed/7540688

1973;**10**(1):33-44

2008;**141**(4):549-556

stemcells.2005-0427

[43] Iwatani M, Ikegami K,

Kremenska Y, Hattori N, Tanaka S, Yagi S, et al. Dimethyl Sulfoxide has an impact on epigenetic profile in mouse Embryoid body. Stem Cells. 2006;**24**(11):2549-2556. Available at: http://doi.wiley.com/10.1634/

[44] Tompkins JD, Hall C, Chen VC-Y, Li AX, Wu X, Hsu D, et al. Epigenetic stability, adaptability, and reversibility in human embryonic stem cells. Proceedings of the National Academy of Sciences of the United States of America. 2012;**109**(31):12544-12549. Available at: http://www.pnas.org/cgi/

doi/10.1073/pnas.1209620109

[45] Yoon SJ, Rahman MS, Kwon WS, Park YJ, Pang MG. Addition of

ultra-low temperatures. Cryobiology.

[31] Karran G, Legge M. Non-enzymatic formation of formaldehyde in mouse oocyte freezing mixtures. Human Reproduction. 1996;**11**(12):2681-2686. Available at: http://www.ncbi.nlm.nih.

Niki E. Cytotoxic effect of formaldehyde with free radicals via increment of cellular reactive oxygen species. Toxicology. 2005;**210**(2-3):235-245

2017;**76**:74-91

gov/pubmed/9021372

1990;**5**(2):212-216

[32] Saito Y, Nishio K, Yoshida Y,

[33] Sommerfeld V, Niemann H. Cryopreservation of bovine in vitro produced embryos using ethylene glycol in controlled freezing or vitrification. Cryobiology. 1999;**38**(2):95-105

[34] Damien M, Luciano AA, Peluso JJ. Propanediol alters intracellular pH and developmental potential of mouse zygotes independently of volume change. Human Reproduction.

[35] Xu X, Liu Y, Cui Z, Wei Y, Zhang L. Effects of osmotic and cold shock on adherent human mesenchymal stem cells during cryopreservation. Journal of

[36] Mullen SF, Agca Y, Broermann DC, Jenkins CL, Johnson CA, Critser JK. The

metaphase II spindle of human oocytes, and the relevance to cryopreservation. Human Reproduction. 2004;**19**(5):

[37] Cole JA, Meyers SA. Osmotic stress stimulates phosphorylation and cellular expression of heat shock proteins in rhesus macaque sperm. Journal of Andrology. 2011;**32**(4):402-410

[38] Christoph K, Beck FX, Neuhofer W.

Osmoadaptation of mammalian cells - an orchestrated network of protective genes. Current Genomics.

Biotechnology. 2012:224-231

effect of osmotic stress on the

*Cryomedia Formula: Cellular Molecular Perspective DOI: http://dx.doi.org/10.5772/intechopen.91382*

ultra-low temperatures. Cryobiology. 2017;**76**:74-91

*Cryopreservation - Current Advances and Evaluations*

post-thaw recovery of peripheral blood stem/progenitor cells using a novel intracellular-like

2009;**11**(4):472-479

1996;**9**:1736-1742

pp. 123-141

cryopreservation solution. Cytotherapy.

[24] Drent M, NAM C, Henderson RF, EFM W, Van Dieijen-Visser M. Usefulness of lactate dehydrogenase and its isoenzymes as indicators of lung damage or inflammation. European Respiratory Journal.

[25] Van BRG. Viability and functional assays used to assess preservation efficacy. In: Baust JG, Baust JM, editors. Advances in Biopreservation. Boca Raton: Taylor & Francis; 2007.

[26] Egea R, Escrivá M, Puchalt N, Varghese A. OMICS: Current and future perspectives in reproductive medicine and technology. Journal of Human Reproductive Sciences. 2014;**7**(2):73. Available at: http://www.jhrsonline.org/

text.asp?2014/7/2/73/138857

[28] Wang S, Wang W, Xu Y,

2014;**14**(2-3):298-310

characteristics of human sperm cryopreservation. Proteomics.

[27] Volk GM. Application of functional genomics and proteomics to plant cryopreservation. Current Genomics. 2010;**11**(1):24-29. Available at: http:// www.pubmedcentral.nih.gov/

articlerender.fcgi?artid=2851113&tool= pmcentrez&rendertype=abstract

Tang M, Fang J, Sun H, et al. Proteomic

[29] Arakawa T, Carpenter JF, Kita YA, Crowe JH. The basis for toxicity of certain cryoprotectants: A hypothesis. Cryobiology. 1990;**27**(4):401-415

[30] Elliott GD, Wang S, Fuller BJ. Cryoprotectants: A review of the actions and applications of cryoprotective solutes that modulate cell recovery from

Simplified molecular biology. Bio/ Technology. 1992;**10**(9):1007-1011

Thermodynamic Mechanism of Protein Stabilization by Trehalose. Biophysical Chemistry. Elsevier; 1997. pp. 25-43

[19] Sampedro JG, Guerra G, Pardo J-P, Uribe S. Trehalose-mediated protection of the plasma membrane H+-ATPase from Kluyveromyces lactis during freezedrying and rehydration. Cryobiology. 1998;**37**(2):131-138. Available at: http:// www.sciencedirect.com/science/article/

[17] Xie G, Timasheff SN. The

[18] Lynch AL, Chen R, NKH S. pH-responsive polymers for trehalose loading and desiccation protection of human red blood cells. Biomaterials. 2011;**32**(19):4443-4449. Available at: http://www.ncbi.nlm.nih.gov/

pubmed/21421265

pii/S0011224098921095

[20] Radaelli MRM, Almodin CG, Minguetti-Câmara VC, Cerialli PMA, Nassif AE, Gonçalves AJ. A comparison between a new vitrification protocol and the slow freezing method in the cryopreservation of prepubertal testicular tissue. JBRA Assisted Reproduction. 2017;**21**(3):188-195

[21] Lee S, Ryu K-J, Kim B, Kang D, Kim YY, Kim T. Comparison between slow freezing and Vitrification for human ovarian tissue cryopreservation and xenotransplantation. International

Journal of Molecular Sciences.

[22] Deller RC, Vatish M, Mitchell DA, Gibson MI. Glycerol-free cryopreservation of red blood cells enabled by ice-recrystallization-inhibiting polymers. ACS Biomaterials Science & Engineering. 2015;**1**(9):789-794. Available at: http://pubs.acs.org/ doi/10.1021/acsbiomaterials.5b00162

[23] Clarke DM, Yadock DJ, Nicoud IB, Mathew AJ, Heimfeld S. Improved

2019;**20**(13):3346

**56**

[31] Karran G, Legge M. Non-enzymatic formation of formaldehyde in mouse oocyte freezing mixtures. Human Reproduction. 1996;**11**(12):2681-2686. Available at: http://www.ncbi.nlm.nih. gov/pubmed/9021372

[32] Saito Y, Nishio K, Yoshida Y, Niki E. Cytotoxic effect of formaldehyde with free radicals via increment of cellular reactive oxygen species. Toxicology. 2005;**210**(2-3):235-245

[33] Sommerfeld V, Niemann H. Cryopreservation of bovine in vitro produced embryos using ethylene glycol in controlled freezing or vitrification. Cryobiology. 1999;**38**(2):95-105

[34] Damien M, Luciano AA, Peluso JJ. Propanediol alters intracellular pH and developmental potential of mouse zygotes independently of volume change. Human Reproduction. 1990;**5**(2):212-216

[35] Xu X, Liu Y, Cui Z, Wei Y, Zhang L. Effects of osmotic and cold shock on adherent human mesenchymal stem cells during cryopreservation. Journal of Biotechnology. 2012:224-231

[36] Mullen SF, Agca Y, Broermann DC, Jenkins CL, Johnson CA, Critser JK. The effect of osmotic stress on the metaphase II spindle of human oocytes, and the relevance to cryopreservation. Human Reproduction. 2004;**19**(5): 1148-1154

[37] Cole JA, Meyers SA. Osmotic stress stimulates phosphorylation and cellular expression of heat shock proteins in rhesus macaque sperm. Journal of Andrology. 2011;**32**(4):402-410

[38] Christoph K, Beck FX, Neuhofer W. Osmoadaptation of mammalian cells - an orchestrated network of protective genes. Current Genomics.

2007;**8**(4):209-218. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/18645598

[39] Meryman HT, Hornblower M. A method for freezing and washing red blood cells using a high glycerol concentration. Transfusion. 1972;**12**(3):145-156

[40] Ogura T, Shuba LM, McDonald TF. Action potentials, ionic currents and cell water in Guinea pig ventricular preparations exposed to dimethyl sulfoxide. Journal of Pharmacology and Experimental Therapeutics. 1995;**273**:1273-1286. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/7540688

[41] Pribor DB, Nara A. The effect of salt or various cryoprotective agents on frog sciatic nerves. Cryobiology. 1973;**10**(1):33-44

[42] Willekens FLA, Werre JM, Groenen-Döpp YAM, Roerdinkholder-Stoelwinder B, De Pauw B, Bosman GJCGM. Erythrocyte vesiculation: A self-protective mechanism? British Journal of Haematology. 2008;**141**(4):549-556

[43] Iwatani M, Ikegami K, Kremenska Y, Hattori N, Tanaka S, Yagi S, et al. Dimethyl Sulfoxide has an impact on epigenetic profile in mouse Embryoid body. Stem Cells. 2006;**24**(11):2549-2556. Available at: http://doi.wiley.com/10.1634/ stemcells.2005-0427

[44] Tompkins JD, Hall C, Chen VC-Y, Li AX, Wu X, Hsu D, et al. Epigenetic stability, adaptability, and reversibility in human embryonic stem cells. Proceedings of the National Academy of Sciences of the United States of America. 2012;**109**(31):12544-12549. Available at: http://www.pnas.org/cgi/ doi/10.1073/pnas.1209620109

[45] Yoon SJ, Rahman MS, Kwon WS, Park YJ, Pang MG. Addition of

cryoprotectant significantly alters the epididymal sperm proteome. PLoS One. 2016;**11**(3):1-15

[46] Chen G, Ren L, Zhang J, Reed BM, Zhang D, Shen X. Cryopreservation affects ROS-induced oxidative stress and antioxidant response in Arabidopsis seedlings. Cryobiology. 2015;**70**(1):38-47. Available at: http:// linkinghub.elsevier.com/retrieve/pii/ S0011224014007068

[47] Gadea J, Sellés E, Marco MA, Coy P, Matás C, Romar R, et al. Decrease in glutathione content in boar sperm after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders. Theriogenology. 2004;**62**(3-4):690-701

[48] Kadirvel G, Kumar S, Kumaresan A. Lipid peroxidation, mitochondrial membrane potential and DNA integrity of spermatozoa in relation to intracellular reactive oxygen species in liquid and frozen-thawed buffalo semen. Animal Reproduction Science. 2009;**114**(1-3):125-134

[49] Peris SI, Bilodeau JF, Dufour M, Bailey JL. Impact of cryopreservation and reactive oxygen species on DNA integrity, lipid peroxidation, and functional parameters in ram sperm. Molecular Reproduction and Development. 2007;**74**(7):878-892

[50] Kanias T, Acker JP. Trehalose loading into red blood cells is accompanied with hemoglobin oxidation and membrane lipid peroxidation. Cryobiology. 2015;**58**(2):232-239. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/19135990

[51] Baumber J, Ball BA, Linfor JJ, Meyers SA. Reactive oxygen species and cryopreservation promote DNA fragmentation in equine spermatozoa. Journal of Andrology. 2003;**24**(4):621-628

[52] Li P, Li Z-H, Dzyuba B, Hulak M, Rodina M, Linhart O. Evaluating the impacts of osmotic and oxidative stress on common carp (Cyprinus carpio, L.) sperm caused by cryopreservation Techniques1. Biology of Reproduction. 2010;**83**(5):852-858. Available at: https://academic.oup.com/ biolreprod/article-lookup/doi/10.1095/ biolreprod.110.085852

[53] Xu X, Cowley S, Flaim CJ, James W, Seymour L, Cui Z. The roles of apoptotic pathways in the low recovery rate after cryopreservation of dissociated human embryonic stem cells. Biotechnology Progress. 2010;**26**(3):827-837

[54] de Boer F, Dräger AM, Pinedo HM, Kessler FL, Monnee-van Muijen M, Weijers G, et al. Early apoptosis largely accounts for functional impairment of CD34+ cells in frozen-thawed stem cell grafts. Journal of Hematotherapy & Stem Cell Research. 2002;**11**(6):951-963. Available at: http://online.liebertpub.com/doi/ abs/10.1089/152581602321080619

[55] Takahashi T, Igarashi H, Doshida M, Takahashi K, Nakahara K, Tezuka N, et al. Lowering intracellular and extracellular calcium contents prevents cytotoxic effects of ethylene glycol-based Vitrification solution in unfertilized mouse oocytes. Molecular Reproduction and Development. 2004;**68**(2):250-258

[56] Kadić E, Moniz RJ, Huo Y, Chi A, Kariv I. Effect of cryopreservation on delineation of immune cell subpopulations in tumor specimens as determinated by multiparametric single cell mass cytometry analysis. BMC Immunology. 2017;**18**(1):6. https://doi. org/10.1186/S12865-017-0192-1

[57] Nynca J, Arnold GJ, Fröhlich T, Ciereszko A. Cryopreservation-induced alterations in protein composition of

**59**

*Cryomedia Formula: Cellular Molecular Perspective DOI: http://dx.doi.org/10.5772/intechopen.91382*

> The Aging Male. 2018. DOI: 10.1080/13685538.2018.1529156

stem cells. Artificial Cells,

2017;**45**(1):63-68

[65] Aliakbari F, Sedighi Gilani MA, Yazdekhasti H, Koruji M, Asgari HR, Baazm M, et al. Effects of antioxidants, catalase and α-tocopherol on cell viability and oxidative stress variables in frozen-thawed mice spermatogonial

Nanomedicine, and Biotechnology.

[66] Taylor K, Roberts P, Sanders K, Burton P. Effect of antioxidant supplementation of cryopreservation medium on post-thaw integrity of human spermatozoa. Reproductive Biomedicine Online. 2009;**18**(2):184-189

[67] Henry L, Fransolet M, Labied S, et al. Supplementation of transport and freezing media with anti-apoptotic

survival. Journal of Ovarian Research. 2016;**9**:4. https://doi.org/10.1186/

drugs improves ovarian cortex

[68] Al-Otaibi NAS, Cassoli JS, Martins-De-Souza D, Slater NKH, Rahmoune H. Human leukemia cells (HL-60) proteomic and biological signatures underpinning cryo-damage are differentially modulated by novel cryo-additives. GigaScience.

S13048-016-0216-0

2018;**8**(3):1-13

rainbow trout semen. Proteomics.

[58] Al-Otaibi NAS. Novel cryo-

[59] Ujihira M, Iwama A, Aoki M, Aoki K, Omaki S, Goto E, et al.

protective agents to improve the quality of cryopreserved mammalian cells [PhD Thesis]. University of Cambridge; 2018

Cryoprotective effect of low-molecularweight hyaluronan on human dermal fibroblast monolayers. Cryo-Letters.

[60] Takeo T, Sztein J, Nakagata N. The CARD method for mouse sperm cryopreservation and in vitro fertilization using frozen-thawed sperm. In: Methods in Molecular Biology. New York, NY: Humana Press;

Nekoonam S, Naji M, Bakhshalizadeh S, Amidi F. Cryoprotective effect of resveratrol on DNA damage and crucial human sperm messenger RNAs, possibly through 5′ AMP-activated protein kinase activation. Cell and Tissue Banking. 2018;**19**(1):87-95

[62] NAS A, NKH S, Rahmoune H. Salidroside as a novel protective agent to improve red blood cell cryopreservation. PLoS One. 2016;**11**(9):e0162748. Available at: http://www.pubmedcentral.nih.gov/ articlerender.fcgi?artid=5025239&tool= pmcentrez&rendertype=abstract

[63] Tomás RMF, Bailey TL, Hasan M, Gibson MI. Extracellular antifreeze protein significantly enhances the cryopreservation of cell monolayers.

[64] Aghaz F, Khazaei M, Vaisi-Raygani A, Bakhtiyari M. Cryoprotective effect of sericin supplementation in freezing and thawing media on the outcome of cryopreservation in human sperm.

Biomacromolecules; 14 Oct 2019;**20**(10):3864-3872

2015;**15**(15):2643-2654

2010;**31**(2):101-111

2019. pp. 243-256

[61] Shabani Nashtaei M,

*Cryomedia Formula: Cellular Molecular Perspective DOI: http://dx.doi.org/10.5772/intechopen.91382*

rainbow trout semen. Proteomics. 2015;**15**(15):2643-2654

*Cryopreservation - Current Advances and Evaluations*

[52] Li P, Li Z-H, Dzyuba B, Hulak M, Rodina M, Linhart O. Evaluating the impacts of osmotic and oxidative stress on common carp (Cyprinus carpio, L.) sperm caused by cryopreservation Techniques1. Biology of Reproduction.

biolreprod/article-lookup/doi/10.1095/

James W, Seymour L, Cui Z. The roles of apoptotic pathways in the low recovery rate after cryopreservation of dissociated human embryonic stem cells. Biotechnology Progress.

[54] de Boer F, Dräger AM, Pinedo HM, Kessler FL, Monnee-van Muijen M, Weijers G, et al. Early apoptosis largely accounts for functional impairment

cells in frozen-thawed

Hematotherapy & Stem Cell Research. 2002;**11**(6):951-963. Available at: http://online.liebertpub.com/doi/ abs/10.1089/152581602321080619

Doshida M, Takahashi K, Nakahara K, Tezuka N, et al. Lowering intracellular and extracellular calcium contents prevents cytotoxic effects of ethylene glycol-based Vitrification solution in unfertilized mouse oocytes. Molecular Reproduction and Development.

[56] Kadić E, Moniz RJ, Huo Y, Chi A, Kariv I. Effect of cryopreservation on delineation of immune cell

subpopulations in tumor specimens as determinated by multiparametric single cell mass cytometry analysis. BMC Immunology. 2017;**18**(1):6. https://doi. org/10.1186/S12865-017-0192-1

[57] Nynca J, Arnold GJ, Fröhlich T, Ciereszko A. Cryopreservation-induced alterations in protein composition of

2010;**83**(5):852-858. Available at: https://academic.oup.com/

[53] Xu X, Cowley S, Flaim CJ,

biolreprod.110.085852

2010;**26**(3):827-837

stem cell grafts. Journal of

[55] Takahashi T, Igarashi H,

2004;**68**(2):250-258

of CD34+

cryoprotectant significantly alters the epididymal sperm proteome. PLoS One.

[46] Chen G, Ren L, Zhang J, Reed BM, Zhang D, Shen X. Cryopreservation affects ROS-induced oxidative stress and antioxidant response in Arabidopsis seedlings. Cryobiology. 2015;**70**(1):38-47. Available at: http:// linkinghub.elsevier.com/retrieve/pii/

[47] Gadea J, Sellés E, Marco MA, Coy P, Matás C, Romar R, et al. Decrease in glutathione content in boar sperm after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders. Theriogenology.

[48] Kadirvel G, Kumar S, Kumaresan A. Lipid peroxidation, mitochondrial membrane potential and DNA

integrity of spermatozoa in relation to intracellular reactive oxygen species in liquid and frozen-thawed buffalo semen. Animal Reproduction Science.

[49] Peris SI, Bilodeau JF, Dufour M, Bailey JL. Impact of cryopreservation and reactive oxygen species on DNA integrity, lipid peroxidation, and functional parameters in ram sperm. Molecular Reproduction and Development. 2007;**74**(7):878-892

[50] Kanias T, Acker JP. Trehalose loading into red blood cells is accompanied with hemoglobin oxidation and membrane lipid peroxidation. Cryobiology. 2015;**58**(2):232-239. Available at: http://www.ncbi.nlm.nih.gov/

[51] Baumber J, Ball BA, Linfor JJ, Meyers SA. Reactive oxygen species and cryopreservation promote DNA fragmentation in equine spermatozoa. Journal of Andrology.

pubmed/19135990

2003;**24**(4):621-628

2016;**11**(3):1-15

S0011224014007068

2004;**62**(3-4):690-701

2009;**114**(1-3):125-134

**58**

[58] Al-Otaibi NAS. Novel cryoprotective agents to improve the quality of cryopreserved mammalian cells [PhD Thesis]. University of Cambridge; 2018

[59] Ujihira M, Iwama A, Aoki M, Aoki K, Omaki S, Goto E, et al. Cryoprotective effect of low-molecularweight hyaluronan on human dermal fibroblast monolayers. Cryo-Letters. 2010;**31**(2):101-111

[60] Takeo T, Sztein J, Nakagata N. The CARD method for mouse sperm cryopreservation and in vitro fertilization using frozen-thawed sperm. In: Methods in Molecular Biology. New York, NY: Humana Press; 2019. pp. 243-256

[61] Shabani Nashtaei M, Nekoonam S, Naji M, Bakhshalizadeh S, Amidi F. Cryoprotective effect of resveratrol on DNA damage and crucial human sperm messenger RNAs, possibly through 5′ AMP-activated protein kinase activation. Cell and Tissue Banking. 2018;**19**(1):87-95

[62] NAS A, NKH S, Rahmoune H. Salidroside as a novel protective agent to improve red blood cell cryopreservation. PLoS One. 2016;**11**(9):e0162748. Available at: http://www.pubmedcentral.nih.gov/ articlerender.fcgi?artid=5025239&tool= pmcentrez&rendertype=abstract

[63] Tomás RMF, Bailey TL, Hasan M, Gibson MI. Extracellular antifreeze protein significantly enhances the cryopreservation of cell monolayers. Biomacromolecules; 14 Oct 2019;**20**(10):3864-3872

[64] Aghaz F, Khazaei M, Vaisi-Raygani A, Bakhtiyari M. Cryoprotective effect of sericin supplementation in freezing and thawing media on the outcome of cryopreservation in human sperm.

The Aging Male. 2018. DOI: 10.1080/13685538.2018.1529156

[65] Aliakbari F, Sedighi Gilani MA, Yazdekhasti H, Koruji M, Asgari HR, Baazm M, et al. Effects of antioxidants, catalase and α-tocopherol on cell viability and oxidative stress variables in frozen-thawed mice spermatogonial stem cells. Artificial Cells, Nanomedicine, and Biotechnology. 2017;**45**(1):63-68

[66] Taylor K, Roberts P, Sanders K, Burton P. Effect of antioxidant supplementation of cryopreservation medium on post-thaw integrity of human spermatozoa. Reproductive Biomedicine Online. 2009;**18**(2):184-189

[67] Henry L, Fransolet M, Labied S, et al. Supplementation of transport and freezing media with anti-apoptotic drugs improves ovarian cortex survival. Journal of Ovarian Research. 2016;**9**:4. https://doi.org/10.1186/ S13048-016-0216-0

[68] Al-Otaibi NAS, Cassoli JS, Martins-De-Souza D, Slater NKH, Rahmoune H. Human leukemia cells (HL-60) proteomic and biological signatures underpinning cryo-damage are differentially modulated by novel cryo-additives. GigaScience. 2018;**8**(3):1-13

**61**

Section 2

Procedures for

Cryopreserving Gametes

Section 2
