**3. Five basic methods of long-term cell biostabilization:** *pro's* **&** *con's*

## **3.1 All five basic methods of long-term biostabilization cell requires vitrification of the intracellular milieu**

We have defined *5 major ways* of cell stabilization that all lead to low- or high-temperature VF of intracellular milieu as we outlined in [Katkov *et al.*, 2006], which are shown on a schematic phase diagram (**Fig. 1**) adapted from [Devireddy & Thirumala, 2011] with some corrections and additions.

Equilibrium (slow) freezing (points A-B' in green) allows to freeze-out the bulk of both extracellular and intracellular water (which escapes from the cell as the extracellular liquid phase becomes more and more concentrated) to ice. Finally, the cells are vitrified in the inter-ice "channels" that are surrounded by ice but always make a connected network (due to barometric restrictions) and surrounded by ice. Yet, the glass transition temperature in those channels is still low so the cells must be stored in LN2 at -196 OC, in nitrogen vapor, or in industrials freezers at -130OC and for a limited time at higher temperature than the *Tg* of water (around -136OC), for example in more accessible -80OC freezers. This is the mainstream conventional cryopreservation, which in the majority of cases requires the use of permeable and impermeable cryoprotective agents (CPAs).

Ice-free *equilibrium* vitrification (E-VF) of cell suspensions, tissues, and organs at very low temperatures and moderate to high rates of freezing (points E-F in red). This method requires the use of high concentrations of vitrificants, which elevates the viscosity of the milieu and prevents the ice formation during cooling and de-vitrification (sometimes called re-crystallization, which is not exactly the same) during warming. Some researchers [Fahy & Rall, 2007] refer to this method as *"vitrification proper",* and in its "pure form" (see below) has had very limited success in preserving animal oocytes, embryos, some tissues and *one* organ, as well as some plant specimens.

*Intracellular* ice-free *kinetic* vitrification of a bulk solution by very fast (abrupt) plunging into a cooling agent such as liquid nitrogen (points G-H in purple). The extremely high rate of cooling (104–106 OC/min) and practically instant warming prevents ice formation inside the cells (the ice still can be formed outside but it has no time to cause any osmotic damage to the cells as K-VF occurs in fractions of a second). As the result, it does not require the use of potentially toxic high concentrations of "CPAs" (vitrificants) or no permeable exogenous vitrificants at all, it is referred to as *"CPA-free vitrification"* by the Isachenkos in regards to sperm. We deliberately include in this method cooling of sperm at much lower rates because the very high *Tg* of the intracellular milieu does not require such high rates. This is one of the major themes of this chapter.

Slow freezing to moderately low (around -40 OC -- -60OC) temperatures, which comprises two steps; i) primary drying - sublimation of the bulk of ice at very high vacuum (points A-D, and ii) secondary drying of the 'cake' at elevated (up to +30-40 OC) temperatures (points D-C). This method is called *lyophilization* and it is widely used in food production, microbiology and in the pharmaceutical industry; but so far it has had very limited applications in the preservation of *animal* cells and higher plants.

Kinetic Vitrification of Spermatozoa of Vertebrates: What Can We Learn from Nature? 11

First, we have to remember that "*successful* (i.e., bringing offspring) freeze-drying or drying of spermatozoa" is a confusing and actually misleading statement. The *properly* freezedried/desiccated spermatozoa are dead, they are never motile, and neither do they have intact acrosome (in the majority of cases). It is the genetic apparatuses, which include such excellent endogenous vitrificants as proteins (e.g., histones), and at lesser extent, DNA that indeed can be stabilized at high temperatures (above 0OC) for long time by xeropreservation (preferably) or by lyophilization (if secondary drying is done properly). Naturally, if intracellular sperm injection (ICSI) is performed, both methods can and do bring offspring

Secondly, as the nucleus of somatic cells can be kept intact after desiccation, it (theoretically) can be used for cloning by somatic-cell intracellular nuclear transfer (SCNT). So, those two aspects, ICSI and SCNT raise the question whether the xerobanks of both gametes and somatic cells should be created for human, model (laboratory), agricultural and wildlife species. We personally believe (though it might change with the time) that except for xerobanks of sperm of laboratory animals, such as transgenic mice and rats, for which both ICSI and SCNT have been well established [Katkov, 2008], the other types of xerobanks are not a necessity, and people should focus their resources and money (which are often scarce in this field) on the methods that have been proven to produce viable cells ( i.e. on cryopreservation). In situations where the cold chain is not as easily available (for example, for the preservation of a genome of species that are on the verge of extinction), drying could be considered as the last resort, but for now, it should not be considered as an alternative to cryobanks. That might change where ICSI and SCNT become routine for many species, but so far we should concentrate on CP. And again, it is gametes, embryos and other reproductive cells that should be preserved first to save genetic material of endangered species even after their death [Maksudov *et al.*, 2009] while, for example, the CP of stem and other somatic cells should be kept as the last resort when the reproductive cells are unavailable. Note, that some other authors of this Book are much more optimistic on that matter of both drying (e.g., the Chapter by Joseph Saragusty ([Saragusty, 2012] sub-chapters 2.3 and 4.2), and cryobanks of stem cells for restoration of species ( [Saragusty, 2012], sub-

**3.2.1 Freeze-drying/desiccation of spermatozoa has not produced** *motile and viable cells* **but it fits for intracellular sperm injection (ICSI) as it stabilizes the nucleus** 

**3.2.2 On reasoning of creating** *"xerobanks"* **of dried genetic material** 

**3.3 Slow freezing: Still the mainstream of cryopreservation but…** 

As we mentioned above, the discovery of "enigmatic glycerol" [Polge *et al.*, 1949; Smirnov, 1949] led to the explosion of methods of cryopreservation and types of species cryopreserved and development of the first cryobanks that marked the 1950's. It revolutionized first the cattle industry, than blood transfusion and many others followed. However, while many of them being successful, the method *per se* remained semi-empirical. However, it has changed with introduction of the 2-factor hypothesis and the equations for the equilibrium slow freezing (minimal intracellular ice formation) by Peter Mazur [Mazur, 1963; Mazur *et al.*, 1972]. Using this truly fundamental approach, Mazur and colleagues in USA and Ian Wilmut in UK were be able to cryopreserve the mouse embryo [Whittingham *et al.*, 1972; Wilmut, 1972]. Since then, slow freezing has been the mainstream of modern cryobiology, and while VF is an

(see [Suzuki, 2006] for references).

chapter 4.3).

High temperature vitrification of a highly dehydrated sample (desiccation) and its stabilization by air/vacuum drying at temperatures above OC is so no ice is formed (points A-C in orange). In some sources, it also erroneously called *"lyopreservation"* [Chakraborty *et al.*, 2011], which is incorrect as *"lyo"* implies sublimation (Greek *luien-* loosing of ice during sublimation (http://dictionary.reference.com/browse/lyo-). In contrast, the Greek word *xero* means *"dry"* (http://dictionary.reference.com/browse/xero-), thus *"xerophile organisms"*, or even the *Xerox* machine! Subsequently, *xeroportective* agents such as trehalose are often used to prevent damage associated with high levels of dehydration when it is used in secondary drying during freeze-drying, and during the whole desiccation cycle. Note that the temperature of drying *Tdr* is *always* above the glass transition temperature of the sample *Tg* (blue curve) for both methods on definition (otherwise, neither sublimation nor evaporation will occur due to extremely high viscosity). For stable storage on another hand, the temperature of storage *Tst* must be *below Tg*, so the conditions of stable drying are following *Tst < Tg < Tdr(f)* (final temeperature of drying). Many papers on drying of biologicals report *Tg* above *Tdr*, which is incorrect (see [Katkov & Levine, 2004] for details and possible explanation of such *"paradox"*). It can explain instability of samples at long storage [Suzuki, 2006] that are often claimed to have *Tg* +60-70 OC while in fact they barely exceed 0OC or fall within the negative range and cannot be long-term stored at ambient temperatures.

The first three methods imply the low temperature and thus, are in the scope of these two books *("cryo"* means cold). Biostabilization above 0OC is often considered as a part of the preservation science and traditionally reported on the cryo-meeting and published in the specialized journals such as "*Cryobiology"*, *"CryoLetters"* and "*Problems of Cryobiology and Cryomedicine* "(a bilingual journal of the Institute for Cryobiology in Kharkov, Ukraine). We deliberately excluded those topics from the scope of our Books as they need special consideration; nonetheless, we will briefly discuss some aspects below.

#### **3.2 At present, desiccation and especially lyophilization can** *not* **be considered as major approaches for biostabilization of** *viable* **cells**

Despite the reports of "successful" xeropreservation and lyophilization of live vertebrate cells from time to time by many groups including prominent cryobiologists since the end of 1940's, with three notable reports of Meryman and the birth of a cow called "*Desicca*"(see [Suzuki, 2006] for an excellent mini-review on the topic), it turned out that neither of the methods to date have proven to produce *stable* and *viable* cells that could be stored for *long* periods of time. It mainly contributes to the fact that even for such good vitrificants, such as proteins, achieving a true high *Tg* coincides with very low water content (in a range of 0.3 g H2O per 1 g dry weight), which apparently is not sustainable by vertebrate cells insofar. Whether the very recent reports by Devireddy and Thirumala [Devireddy & Thirumala, 2011] and by the Mehmet Toner's group [Chakraborty *et al.*, 2011]will change the situation, or they will fade away as all the proposed methods have so far needs to be seen. Our approach is expressed in [Katkov & Levine, 2004; Katkov *et al.*, 2006; Katkov, 2008]. The discussion of what has been done wrong so far, and what could and should be done, would need a separate Chapter, and as we said before, is out of the scope of this Book. However, there are two things that should be mentioned.

High temperature vitrification of a highly dehydrated sample (desiccation) and its stabilization by air/vacuum drying at temperatures above OC is so no ice is formed (points A-C in orange). In some sources, it also erroneously called *"lyopreservation"* [Chakraborty *et al.*, 2011], which is incorrect as *"lyo"* implies sublimation (Greek *luien-* loosing of ice during sublimation (http://dictionary.reference.com/browse/lyo-). In contrast, the Greek word *xero* means *"dry"* (http://dictionary.reference.com/browse/xero-), thus *"xerophile organisms"*, or even the *Xerox* machine! Subsequently, *xeroportective* agents such as trehalose are often used to prevent damage associated with high levels of dehydration when it is used in secondary drying during freeze-drying, and during the whole desiccation cycle. Note that the temperature of drying *Tdr* is *always* above the glass transition temperature of the sample *Tg* (blue curve) for both methods on definition (otherwise, neither sublimation nor evaporation will occur due to extremely high viscosity). For stable storage on another hand, the temperature of storage *Tst* must be *below Tg*, so the conditions of stable drying are following *Tst < Tg < Tdr(f)* (final temeperature of drying). Many papers on drying of biologicals report *Tg* above *Tdr*, which is incorrect (see [Katkov & Levine, 2004] for details and possible explanation of such *"paradox"*). It can explain instability of samples at long storage [Suzuki, 2006] that are often claimed to have *Tg* +60-70 OC while in fact they barely exceed 0OC or fall within the negative range and cannot be long-term stored at ambient

The first three methods imply the low temperature and thus, are in the scope of these two books *("cryo"* means cold). Biostabilization above 0OC is often considered as a part of the preservation science and traditionally reported on the cryo-meeting and published in the specialized journals such as "*Cryobiology"*, *"CryoLetters"* and "*Problems of Cryobiology and Cryomedicine* "(a bilingual journal of the Institute for Cryobiology in Kharkov, Ukraine). We deliberately excluded those topics from the scope of our Books as they need special

**3.2 At present, desiccation and especially lyophilization can** *not* **be considered as** 

Despite the reports of "successful" xeropreservation and lyophilization of live vertebrate cells from time to time by many groups including prominent cryobiologists since the end of 1940's, with three notable reports of Meryman and the birth of a cow called "*Desicca*"(see [Suzuki, 2006] for an excellent mini-review on the topic), it turned out that neither of the methods to date have proven to produce *stable* and *viable* cells that could be stored for *long* periods of time. It mainly contributes to the fact that even for such good vitrificants, such as proteins, achieving a true high *Tg* coincides with very low water content (in a range of 0.3 g H2O per 1 g dry weight), which apparently is not sustainable by vertebrate cells insofar. Whether the very recent reports by Devireddy and Thirumala [Devireddy & Thirumala, 2011] and by the Mehmet Toner's group [Chakraborty *et al.*, 2011]will change the situation, or they will fade away as all the proposed methods have so far needs to be seen. Our approach is expressed in [Katkov & Levine, 2004; Katkov *et al.*, 2006; Katkov, 2008]. The discussion of what has been done wrong so far, and what could and should be done, would need a separate Chapter, and as we said before, is out of the scope of this Book. However,

consideration; nonetheless, we will briefly discuss some aspects below.

**major approaches for biostabilization of** *viable* **cells** 

there are two things that should be mentioned.

temperatures.

#### **3.2.1 Freeze-drying/desiccation of spermatozoa has not produced** *motile and viable cells* **but it fits for intracellular sperm injection (ICSI) as it stabilizes the nucleus**

First, we have to remember that "*successful* (i.e., bringing offspring) freeze-drying or drying of spermatozoa" is a confusing and actually misleading statement. The *properly* freezedried/desiccated spermatozoa are dead, they are never motile, and neither do they have intact acrosome (in the majority of cases). It is the genetic apparatuses, which include such excellent endogenous vitrificants as proteins (e.g., histones), and at lesser extent, DNA that indeed can be stabilized at high temperatures (above 0OC) for long time by xeropreservation (preferably) or by lyophilization (if secondary drying is done properly). Naturally, if intracellular sperm injection (ICSI) is performed, both methods can and do bring offspring (see [Suzuki, 2006] for references).
