**3.2 Usage of different cryoprotective agents and vehicles**

During the cryopreservation course, as the cell suspension is cooled below the freezing point, ice crystals form and the concentration of the solutes in the suspension increases, being both processes damaging for the cells. Cryoprotective agents (CPAs) are necessary to minimize or prevent the damage associated with the freezing process. The mechanisms providing this protection in slow cooling-rapid thawing protocols, although not completely understood, appear to work primarily by altering the physical conditions of both the ice and the solutions immediately surrounding (external to) the cells. In contrast, vitrification overcomes the problems associated with ice crystallization in a different manner. Here cryoprotectants are used in high concentrations preventing ice formation entirely (Baust et al., 2009; Hunt, 2011).

Different CPAs have been identified so far to be used for the cryopreservation of mammalian cells (Klebe & Mancuso, 1983; Matsumura et al., 2010); however, the two most commonly used substances are glycerol and DMSO. Other substances used include sugars, polymers, alcohols and proteins. CPAs can be divided roughly into two different categories: (1) permeating CPAs: substances that permeate the cell membrane (e.g. DMSO and glycerol); and (2) nonpermeating CPAs: impermeable substances (e.g. polyethylenen glycol and trehalose); both types present a different impact on the freezing process. Permeating CPAs have a low molecular weight and thus can penetrate the cell membrane and gradually substitute the water present in the cells. The osmolality of the cells is thereby increased, and subsequently, the percentage of extracellular water that can form ice crystals before reaching the osmotic equilibrium is reduced and total dehydration of the cells is prevented. Nonpermeating CPAs cannot penetrate the cell membrane and stabilize the cell by forming a viscous glassy shell around its surface (Hubel, 1997; Karlsson, 2002; Karlsson & Toner, 1996; Meryman, 2007). Therefore, the selection of an appropriate CPA or a combination of them used in optimal concentrations within an effective vehicle solution will determine the efficiency of the cryopreservation process for a given cell type. In this sense, alternatives to DMSO as the cryoprotectant of choice for hPSCs have been tested due to the known effect of this solvent on inducing differentiation and cytotoxicity (Adler et al., 2006). See Table 1 for an overview of CPAs and freezing vehicles used in different protocols describe here.

improvement of the cryopreservation technique presents a similar yield of hESCs recovery after thawing with low differentiation rates comparable with the results of Reubinoff et al (Reubinoff et al., 2001). The usage of cryovials for vitrification has also been explored showing interesting results. Nishigaki et al used a DMSO-based and serum-free vitrification medium to cryopreserve iPSCs in cryovials (Nishigaki et al., 2011). They compared various vitrification solutions containing different concentrations of DMSO, ethylene glycol and polyethylene glycol with knockout serum replacement (KSR) in both DMEM and Euro-collins vehicle solutions. Analysis of the thermal properties of the cryopreservation solutions during the cooling process by differential scanning calorimetry (DSC) indicated that they would vitrify at an optimal cooling rate of ~ -125 ºC/min. Recovery rates between 20-30% are described one day after thawing using 40% ethylene glycol and 10% polyethylene glycol in Euro-Collins solution. Furthermore, cryopreserved cells express undifferentiation markers and keep pluripotency (Nishigaki et al., 2011). Therefore, using this protocol the vitrification of large

During the cryopreservation course, as the cell suspension is cooled below the freezing point, ice crystals form and the concentration of the solutes in the suspension increases, being both processes damaging for the cells. Cryoprotective agents (CPAs) are necessary to minimize or prevent the damage associated with the freezing process. The mechanisms providing this protection in slow cooling-rapid thawing protocols, although not completely understood, appear to work primarily by altering the physical conditions of both the ice and the solutions immediately surrounding (external to) the cells. In contrast, vitrification overcomes the problems associated with ice crystallization in a different manner. Here cryoprotectants are used in high concentrations preventing ice formation entirely (Baust et al., 2009; Hunt, 2011). Different CPAs have been identified so far to be used for the cryopreservation of mammalian cells (Klebe & Mancuso, 1983; Matsumura et al., 2010); however, the two most commonly used substances are glycerol and DMSO. Other substances used include sugars, polymers, alcohols and proteins. CPAs can be divided roughly into two different categories: (1) permeating CPAs: substances that permeate the cell membrane (e.g. DMSO and glycerol); and (2) nonpermeating CPAs: impermeable substances (e.g. polyethylenen glycol and trehalose); both types present a different impact on the freezing process. Permeating CPAs have a low molecular weight and thus can penetrate the cell membrane and gradually substitute the water present in the cells. The osmolality of the cells is thereby increased, and subsequently, the percentage of extracellular water that can form ice crystals before reaching the osmotic equilibrium is reduced and total dehydration of the cells is prevented. Nonpermeating CPAs cannot penetrate the cell membrane and stabilize the cell by forming a viscous glassy shell around its surface (Hubel, 1997; Karlsson, 2002; Karlsson & Toner, 1996; Meryman, 2007). Therefore, the selection of an appropriate CPA or a combination of them used in optimal concentrations within an effective vehicle solution will determine the efficiency of the cryopreservation process for a given cell type. In this sense, alternatives to DMSO as the cryoprotectant of choice for hPSCs have been tested due to the known effect of this solvent on inducing differentiation and cytotoxicity (Adler et al., 2006). See Table 1 for

an overview of CPAs and freezing vehicles used in different protocols describe here.

amounts of cells is feasible and avoids the risk of contamination.

**3.2 Usage of different cryoprotective agents and vehicles** 

Trehalose is a natural disaccharide that has been selected as an attractive CPA for several reasons. First of all, it has been shown to be effective in mammalian cell stabilization at low temperatures and water contents. Secondly, trehalose preserves cell viability by different mechanisms than DMSO (Crowe et al., 2001; Sum et al., 2003; Sum & de Pablo, 2003). Finally, trehalose addition to the cryopreservation medium containing DMSO and fetal bovine serum (FBS) has been proven to increase the viability of hematopoietic precursor cells from 7% to 20% and improved membrane integrity in cryopreserved fetal skin cells (Erdag et al., 2002; Zhang et al., 2003). Ji et al showed that trehalose loading into adherent colonies of hESCs prior to cryopreservation results in small, but significant improvements in cell viability when combined with DMSO treatment and high FBS concentrations (Ji et al., 2004). In the same line of results, it has been demonstrated that trehalose addition to the freezing and post-thawing medium of hESC colonies cryopreserved in suspension in freezing medium containing 10% DMSO, increased the recovery rate by ~3 folds (from 15 to 48%) (Wu et al., 2005). These results suggested that the protective mechanism of trehalose addition might be the reduction of osmotic changes during the freezing and thawing process, although this hypothesis has not been demonstrated. The addition of trehalose did not affect the normal karyotype of the cells neither their pluripotency capacity tested by teratoma formation (Wu et al., 2005).

A comparison between four different types of CPAs for iPSCs cryopreservation has recently been described: DMSO, ethylene glycol, propylene glycol and glycerol (Katkov et al., 2011). Interestingly, the toxicity of these four CPAs was analyzed after 30 minutes exposure of a 10% CPA solution at 37ºC. The results showed that DMSO was the most toxic CPA for iPSCs while glycerol was the least harmful being the other two CPAs in between. Surprisingly, the protective effect exerted by the same CPAs after cryopreservation of small iPSC clumps by the slow cooling-rapid thawing protocol was the opposite, being DMSO the most protective CPA together with ethylene glycol while glycerol was the least protective one. The same result was obtained when iPSCs previously dissociated with AccutaseTM were cryopreserved in the presence of a ROCK inhibitor in combination with the previous mentioned CPAs. Therefore, ethylene glycol was selected as the cryoprotectant of choice since it presents less toxicity than DMSO and exerts similar levels of protection (Katkov et al., 2011). In addition, these results give clear evidence that the low hPSCs recovery rate obtained after cryopreservation is mainly caused by the freezing-thawing procedure, rather than by the process of CPA addition/removal.

The combination of different CPAs has also been tested in comparison to the conventional freezing solution containing 10% DMSO in slow cooling-rapid freezing protocols. Ha et al performed a detailed study about the composition of the cryopreservation medium, initially analyzing the impact of both DMSO and FBS concentration in hESCs recovery (Ha et al., 2005). They reached the conclusion that a combination of 5% DMSO plus 50% FBS was the most effective one sustaining survival rates of 10%. Afterwards, they used this freezing medium composition as a starting point to test different concentrations of other CPAs such as ethylene glycol or glycerol. An increase of 3 fold in the survival rate (around 30%) was obtained when using a combination of 5% DMSO + 50% FBS +10% ethylene glycol that was selected as the most effective cryopreservation medium. Three passages after thawing cryopreserved hESCs retained the key properties and characteristics of hPSCs (Ha et al., 2005).

Cryopreservation of Human Pluripotent Stem Cells: Are We Going in the Right Direction? 149

hESCs Single

colonies

Cells

Colony clumps

clumps

Table 1. Cryoprotectant agents and freezing vehicles used for the cryopreservation of hPSCs using the slow-freezing rapid-thawing protocol. The conditions and recovery rates showed in the table correspond to the best condition tested in the referenced works. (DMSO:

Hydrosyethylstarch; FBS: fetal bovine serum; FCS: fetal calf serum; KSR: Knockout serum replacement; MEF: mouse embryonic fibroblasts). The recovery rates were determined using different tests: (1) % cell recovery determined by QUANTA Coulter Counter measurement of Calcein-PM+/7AAD-. (2) % viability determined with FDA/EB staining at different time points after thawing. (3) Number of colonies 10 days after plating. (4) Cell viability was determined counting the number of cells by the Trypan blue exclusion method immediately after thawing. (5) Cell viability was determined by FACS using propidium iodide staining immediately after thawing. (6) Number of colonies at day 5/total colonies replated. (7) Flow cytometry analysis of apoptotis using Anexin V and propidium iodide immediately after thawing. (8) Fold increase in the number of recovered cells determined using a Z2 Coulter Counter and Size analyzer 4 days after thawing. Fold increase in the number of colonies determined by microscopy. (9) Recovery rate was calculated as follows: the amount of Grade A+B colonies at day 7 post-thawing versus the amount of frozen Grade A+B colonies. (10) Number of colonies 7 days after thawing. (11) MTT assay to measure % survival rate 24 h post-thawing. (12) Viability determined by Trypan blue exclusion method of adherent colonies at different time points after thawing. (13) Recovery was calculated as the number of cells one week after thawing divided by the number of cells at the time of freezing. The recoveries of hPSCs frozen using microcarriers are normalized to the recoveries of hPSCs frozen as free colonies. (14) Cell viability was measured by MTT assay or Alamar Blue assay several days after thawing. (15) Viability or percentage of surviving cells was calculated as a ratio between live hPSCs after thawing and total number of initially frozen cells. Cells were counted using the Trypan blue exclusion method. (16) Recovery rates were estimated as the % of attached and undifferentiated clumps counted 7-8 days after-thawing respect to the initially frozen. (17) Viability was assessed counting the number of colonies stained for

A new cryopreservation formula containing 7.5% DMSO plus 2.5% polyethylene glycol was analyzed in another work (Xu et al., 2010b). This study resulted in slight but significant increase in the hESCs recovery determined by counting the number of cells or colonies in

MEF feeder layer and feeder-free culture

MEF feeder layer and feeder-free culture

Human foreskin feeder layer

MEF feeder layer

25% viability increase in respect to DMSO alone(14)

80-90%

90-96% viability(15)

30% colony recovery (3)

survival(5) (Xu et al.,

(Ji et al., 2004)

2010b)

(Holm et al., 2010)

(Ha et al., 2005)

No hESCs Adherent

hESCs and iPSCs

No hESCs Colony

dimethyl sulfoxide; EG: ethylene glycol; PEG: polyethylene glycol; HES:

10% DMSO+ 35 mM Trehalose

7.5% DMSO 2.5% PEG

10% DMSO + another undisclosed CPA

> 5% DMSO 10% EG

alkaline phosphatase.

40% Growth medium + 50% FBS

Growth medium

Phosphate

50%FBS and DMEM/ F12

buffer No

ROCK inhibitor + P53 inhibitor


**Cell processing**

Colony clumps and single cells

colonies

clumps

Single cells

clumps

Colony clumps

Adherent colonies (microcarriers)

clumps

clumps

hESCs Single cells

hESCs Adherent colonies

**Type of** 

MEF feeder layer

MEF feeder layer

Human foreskin feeder layer

MEF feeder layer and feeder-free culture

MEF feeder layer and feeder-free culture

culture

MEF feeder layer

MEF feeder layer

MEF feeder layer

MEF feeder layer and feeder-free culture

MEF feeder layer

MEF feeder layer

Single cells Feeder-free

Single cells Feeder-free

**culture Recovery rate Reference** 

~20-50%

~60% recovery (1)

60% (30 min) <10% (24 h) (2)

0-55% recovery (16)

50-60% survival(4)

7-8 fold increase in the number of recovered cells or colonies (8)

> ~80% survival(5)

90% viable cells(4)

8-53% survival(17)

85-95% survival(6)

culture 53-65% (7) (Li et al.,

~98% (5 min) 20-30% (90 min) (12)

1.5-1.9 fold increase in recovery rate(13)

~80% recovery (9)

37-48% recovery(10)

recovery(1) (Katkov et

al., 2011)

(Wagh et al., 2011)

(Li et al., 2010b)

(Martin-Ibanez et al., 2008)

(Claassen et al., 2009)

(Xu et al., 2010a)

(Mollamoh ammadi et al., 2009)

(Lee et al., 2010)

(Li et al., 2008b)

2009)

al., 2007)

(Heng et al., 2005)

(Nie et al., 2009)

(T'joen et al., 2011)

(Zhang et al., 2003)

18.7% (11) (Heng et

**Cell type** 

iPSCs

No Adherent

No hESCs Colony

hESCs

hESCs and iPSCs

hESCs and iPSCs

No hESCs Colony

**CPA composition** 

10% DMSO

5% DMSO 5% HES

10% DMSO + 0.2 mol/l Trehalose

10 % EG Not

**Freezing vehicle** 

described

Growth medium

**Addition of other molecules**

ROCK inhibitor

ROCK inhibitor

ROCK inhibitor + P53 inhibitor

inhibitor

ROCK

Z-VAD-FMK

No

inhibitor hESCs

No hESCs

90% KSR No hESCs Colony

No hESCs Colony

90% FCS ROCK

90% KSR

90% (DMEM/F 12 + 20% FBS)

60 % growth medium + 30%FBS

80% (DMEM/ F12 + 20% KSR)


Table 1. Cryoprotectant agents and freezing vehicles used for the cryopreservation of hPSCs using the slow-freezing rapid-thawing protocol. The conditions and recovery rates showed in the table correspond to the best condition tested in the referenced works. (DMSO: dimethyl sulfoxide; EG: ethylene glycol; PEG: polyethylene glycol; HES:

Hydrosyethylstarch; FBS: fetal bovine serum; FCS: fetal calf serum; KSR: Knockout serum replacement; MEF: mouse embryonic fibroblasts). The recovery rates were determined using different tests: (1) % cell recovery determined by QUANTA Coulter Counter measurement of Calcein-PM+/7AAD-. (2) % viability determined with FDA/EB staining at different time points after thawing. (3) Number of colonies 10 days after plating. (4) Cell viability was determined counting the number of cells by the Trypan blue exclusion method immediately after thawing. (5) Cell viability was determined by FACS using propidium iodide staining immediately after thawing. (6) Number of colonies at day 5/total colonies replated. (7) Flow cytometry analysis of apoptotis using Anexin V and propidium iodide immediately after thawing. (8) Fold increase in the number of recovered cells determined using a Z2 Coulter Counter and Size analyzer 4 days after thawing. Fold increase in the number of colonies determined by microscopy. (9) Recovery rate was calculated as follows: the amount of Grade A+B colonies at day 7 post-thawing versus the amount of frozen Grade A+B colonies. (10) Number of colonies 7 days after thawing. (11) MTT assay to measure % survival rate 24 h post-thawing. (12) Viability determined by Trypan blue exclusion method of adherent colonies at different time points after thawing. (13) Recovery was calculated as the number of cells one week after thawing divided by the number of cells at the time of freezing. The recoveries of hPSCs frozen using microcarriers are normalized to the recoveries of hPSCs frozen as free colonies. (14) Cell viability was measured by MTT assay or Alamar Blue assay several days after thawing. (15) Viability or percentage of surviving cells was calculated as a ratio between live hPSCs after thawing and total number of initially frozen cells. Cells were counted using the Trypan blue exclusion method. (16) Recovery rates were estimated as the % of attached and undifferentiated clumps counted 7-8 days after-thawing respect to the initially frozen. (17) Viability was assessed counting the number of colonies stained for alkaline phosphatase.

A new cryopreservation formula containing 7.5% DMSO plus 2.5% polyethylene glycol was analyzed in another work (Xu et al., 2010b). This study resulted in slight but significant increase in the hESCs recovery determined by counting the number of cells or colonies in

Cryopreservation of Human Pluripotent Stem Cells: Are We Going in the Right Direction? 151

cryopreserved as small clumps (Li et al., 2008b). In parallel our group reported that dissociated hESCs could be cryopreserved in the presence of ROCK inhibitor (Martin-Ibanez et al., 2008; Martin-Ibanez et al., 2009). The addition of Y-27632 to the freezing medium did not increase the formation of hESCs colonies compared to the control non treated cells although it increased cell survival. In contrast, the presence of ROCK inhibitor in the post-thawing recovery medium did increase the formation of hESCs colonies significantly (50-100 times). The addition of Y-27632 to both, the cryopreservation and the post-thawing medium was the condition tested contributing to the highest cell recovery after freezing.

> **Recovery rate (method of analysis)**

50-60% survival % survival after thawing analyzed by Trypan Blue exclusion method

60-80% viable cells % survival after thawing analyzed by Trypan blue exclusion method

80-90% survival % survival after thawing analyzed by propidium iodide staining

20-60% recovery depending on the type of culture % recovery after thawing by QUANTA Coulter Counter measurement of Calcein-PM+/7AAD-

85-95% survival Number of colonies at day 5/total colonies replated

53-65% Flow citometry analysis of apoptosis using Anexin V and propidium iodide

7-8 fold increase in the number of recovered cells 4 days after thawing (Z2 Coulter Counter and Size analyzer)

7 fold increase in the number of colonies 48 h post-thawing

Table 2. Overview of the ROCK inhibitor treatments tested to improve the recovery rates after cryopreservation of hPSCs. Survival rates showed in the table are obtained using the

**Time of** 

Immediately after thawing

Immediately after thawing

Immediately after thawing

5 days after thawing

Immediately after thawing and 24 h after thawing

4 days after thawing

2 days after thawing

**analysis Reference** 

Not stated (Katkov et

(Martin-Ibanez et al., 2008)

(Mollamoh ammadi et al., 2009)

(Xu et al., 2010a) (Xu et al., 2010b)

al., 2011)

(Li et al., 2008b)

(Li et al., 2009)

(Claassen et al., 2009)

(Claassen et al., 2009)

**Rock inhibitor addition** 

Freezing and postthawing recovery media (1 day)

Postthawing recovery medium (1 day)

Postthawing recovery medium (4 days)

Postthawing recovery medium (2 days)

**Type of cell and culture** 

> hESCs on human foresking feeders

hESCs and iPSCs in feeder-free culture

hESCs on MEF feeders and feederfree culture

iPSCs on MEF feeders

hESCs on MEF feeders

hESCs in feeder-free culture

hESCs on MEF feeders and feederfree culture

iPSCs on

best condition tested in the work referenced.

MEF feeders Single cells

**Cell processing** 

Single cells

Single cells

Single cells

Single cells, clumps and adherent colonies

> Colony clumps

Single cells

Single cells

feeder-independent or feeder-dependent culture respectively (Xu et al., 2010b). Recently, an alternative cryopreservation medium combining intracellular (5% DMSO) and extracellular (5% Hydrosyethylstarch) CPAs has been proven to be highly effective for the cryopreservation of small hESC clumps by the classical slow-freezing rapid-thawing method. These clumps are obtained by a combination of hESC colony detachment with Collagenase IV followed by 5 minutes dissociation using an undisclosed solution. This protocol is suitable for handling bulk amounts of hPSCs (T'joen et al., 2011).

Comparison of different freezing vehicles using DMSO as a cryoprotectant has also been studied for the cryopreservation of dissociated hESCs (Mollamohammadi et al., 2009). Three preservation media containing 10% DMSO plus: 90% fetal calf serum (FCS), 90% KSR or 90% hESCs medium containing 20% KSR and ROCK inhibitor were analyzed. The percentage of viable cells obtained by the Trypan blue exclusion method after thawing showed that cells were better preserved in the presence of 90% FCS as a vehicle (~90%). The other two freezing solutions caused lower survival rates (60-80%) (Mollamohammadi et al., 2009). Following a similar approach, Ha et al studied the impact of different FBS concentrations (5, 50 and 95%) in the vehicle freezing solution using a 5% DMSO as a CPA (Ha et al., 2005). A decrease in the survival rate is observed as the FBS concentration is reduced although no differences were found between 50 and 95%. Therefore, the authors established 50% of FBS as the optimal concentration to support hPSCs survival during the cryopreservation process (Ha et al., 2005).
