**3.4 Cryopreservation of adherent versus suspension hPSC colonies**

In view of the poor survival rates obtained after cryopreservation of hPSCs in suspension using the slow-cooling rapid thawing method, some authors decided to test cryopreservation of adherent cells. This decision was based on previous studies done with certain cell types difficult to preserve. For example, hepatocytes cryopreserved in alginate gels display a higher viability and lower apoptotic activity than hepatocytes cryopreserved in suspension (Mahler et al., 2003). Similarly, hepatocytes sandwiched between two layers of collagen provide enhanced viability and protein secretion compared with cells preserved in solution (Birraux et al., 2002; Koebe et al., 1990; Koebe et al., 1999). Taking these results as a proof of principle, hESCs were successfully cryopreserved as adherent colonies in 24 well plates in medium containing 10% DMSO + 30% FBS by Ji et al (Ji et al., 2004). This approach demonstrated that hESCs frozen as adherent colonies were five times more viable than clumps of colonies frozen in suspension. In addition, encapsulation of hESCs colonies inside MatrigelTM for 1 or 2 days increased viability significantly respect to unencapsulated adherent frozen colonies or colonies encapsulated for just 1 h. The percentage of adherent hESC colonies recovered 1 to 2 weeks after cryopreservation was about 80-90% and almost no differentiation was detected. In

Moreover, we described a complete avoidance of hESCs differentiation just after cryopreservation showing that most of the colonies expressed the undifferentiation markers: Oct-4, nanog, SSEA-4, TRA-1-81 and TRA-1-60. The addition of Y-27632 increased the growth rates to control levels, did not affect hESCs normal karyotype and kept their pluripotency (Martin-Ibanez et al., 2008). Similar results have been shown not only for hESCs but also for iPSCs in both feeder-associated and feeder-free conditions (Claassen et al., 2009; Katkov et al., 2011; Mollamohammadi et al., 2009). See table 2 for a sum up of all

ROCK inhibitors have also been used in combination with other molecules such as Caspase inhibitors, p53 inhibitors or Bax inhibitors added always to the post-thawing culture medium. Xu et al showed that none of the three combinations pan-Caspase inhibitors + Y-27632, Caspase 9 inhibitor + Y-27632 and Bax inhibitor + Y-27632 enhanced the protective effect of ROCK inhibitor alone for cryopreserved hESCs (Xu et al., 2010a). Only the treatment with a p53 inhibitor + Y27632 induced a cell recovery similar to that of ROCK inhibitor. However, treatment with p53 alone did not account for an increase in cell survival (Xu et al., 2010a). Similar results were obtained by the same group in another report where they observed an enhancement of hESCs recovery when cryopreserved in 10% DMSO or 7.5% DMSO + 2.5% polyethylene glycol and treated with p53 inhibitor + Y-27632 in the post-

Although most of the works studying the effect of ROCK inhibitor during cryopreservation did not address the mechanism of action of this molecule, at least two of them showed some interesting results (Li et al., 2009; Xu et al., 2010a). Both of them reported a reduction in hESCs apoptosis and/or Caspase activity one day after cryopreservation driven by Y-27632. This is in agreement with the previous report of Watanabe et al who pointed to an antiapoptotic role of this ROCK inhibitor (Watanabe et al., 2007). In addition, Li et al demonstrated that Y-27632 treatment increased the adherent properties of cryopreserved hESCs favoring cell aggregate formation and adhesion to the substrate. This effect, in turn, prevented anoikis and enhanced

In view of the poor survival rates obtained after cryopreservation of hPSCs in suspension using the slow-cooling rapid thawing method, some authors decided to test cryopreservation of adherent cells. This decision was based on previous studies done with certain cell types difficult to preserve. For example, hepatocytes cryopreserved in alginate gels display a higher viability and lower apoptotic activity than hepatocytes cryopreserved in suspension (Mahler et al., 2003). Similarly, hepatocytes sandwiched between two layers of collagen provide enhanced viability and protein secretion compared with cells preserved in solution (Birraux et al., 2002; Koebe et al., 1990; Koebe et al., 1999). Taking these results as a proof of principle, hESCs were successfully cryopreserved as adherent colonies in 24 well plates in medium containing 10% DMSO + 30% FBS by Ji et al (Ji et al., 2004). This approach demonstrated that hESCs frozen as adherent colonies were five times more viable than clumps of colonies frozen in suspension. In addition, encapsulation of hESCs colonies inside MatrigelTM for 1 or 2 days increased viability significantly respect to unencapsulated adherent frozen colonies or colonies encapsulated for just 1 h. The percentage of adherent hESC colonies recovered 1 to 2 weeks after cryopreservation was about 80-90% and almost no differentiation was detected. In

the ROCK inhibitor treatments used for cryopreservation of hPSCs.

hESCs survival (Li et al., 2009; Mollamohammadi et al., 2009).

**3.4 Cryopreservation of adherent versus suspension hPSC colonies** 

thawing medium (Xu et al., 2010b).

contrast, less than 2% of hESC colonies attached when frozen in suspension. A recent work by Katkov et al reported a refinement of the technique cryopreserving adherent iPSC colonies in the presence of ethylene glycol as a cryoprotectant and using a six-step programmed protocol (Katkov et al., 2011). Preservation of iPSCs under these conditions induced a six-fold increase in cell recovery after thawing respect the standard cryopreservation of cell clumps by the slowfreezing rapid thawing method (Katkov et al., 2011). Two mechanisms are postulated to explain the increased viability obtained preserving hESCs as adherent colonies. The first is that hESC colonies do not have to settle to the surface and attach. This is a decisive process for the survival of hPSC colonies frozen in suspension that is rarely achieved due to the massive cell death or cell damage experienced within the colony during cryopreservation. Second, the maintenance of a continuous extracellular matrix signaling may also play a role in the enhanced viability and reduced differentiation of hESCs cryopreserved in an adherent state (Ji et al., 2004). The disadvantage of this technique is that large scale storage is not feasible because hPSCs attached to plates cannot be stored at high density. In addition, culture plates are unable to be sealed like cryovials, increasing the risk of sample cross-contamination during storage in liquid nitrogen. However, methodologies such as preservation on microcarriers might provide the advantages of freezing adherent cells at higher densities that are not possible on flat surfaces. This is what has been described by Nie et al, who used Cytodex 3 microcarriers to cryopreserve adherent hESCs (Nie et al., 2009). These microcarriers consisted of a thin layer of denatured collagen covalently coupled to a matrix of cross-linked dextran. They were modified with MatrigelTM or irradiated MEF to enhance the adhesion of hESC colonies. In this work it was first demonstrated that hESCs colonies were effectively expanded in a pluripotent, undifferentiated state on both types of microcarriers (MatrigelTM and MEF coated). Then cryopreservation utilizing this system was compared to standard freezing of hESC colonies in suspension. hESCs-microcarriers were suspended in freezing medium consisting in 10%DMSO and 30%FBS at a cell density of 1x106 cells/ml on 10 cm2 microcarriers. The suspension was transferred to cryovials, frozen inside a freezing container at a cooling rate of -1ºC/min and moved into liquid nitrogen. Seven days after thawing viability was assessed by counting the number of cells. This number was compared to that of the conventional hESCs slow freezing method. Cryopreservation on microcarriers resulted in 1.7 times the recovery of hESCs frozen in free suspension (Nie et al., 2009). Although the enhancement of cell recovery is not very promising, further optimization of this methodology holds a great potential for future larger-scale cryopreservation.

#### **3.5 Cryopreservation of dissociated single hPSCs versus clumps of colonies**

hPSCs are colony-forming social cells that present a high vulnerability to apoptosis upon cellular detachment and dissociation (Amit et al., 2000; Watanabe et al., 2007). These characteristics could explain why most of the cryopreservation protocols rely on hPSCs small clumps to improve survival rates (Heng et al., 2006; Reubinoff et al., 2001; Richards et al., 2004; Zhou et al., 2004). However, the cryopreservation of clumps presents some associated problems such as limitations on cryoprotectant exposure inside the clump. In this sense, T'Joen et al demonstrated that the application of a cell dissociation solution before freezing, thereby creating a mixed population of very small hESC clumps and single cells, increased the recovery rate after cryopreservation (T'joen et al., 2011). In addition, the use of hPSC colony clumps also prevents a good estimation of freezing-thawing efficiency, as precise cell numbers cannot be estimated. Therefore, development of cryopreservation protocols for dissociated hPSCs is a pre-requisite for the widespread use of these cells in basic or clinical research.

Cryopreservation of Human Pluripotent Stem Cells: Are We Going in the Right Direction? 155

results of this study identified three critical factors for successful hESCs freezing: ice crystal seed at some point above the temperature of spontaneous intracellular ice formation (between -7ºC and -12ºC), an appropriate freezing rate (between -0,3ºC and -1,8ºC/min) and rapid thawing (at 25-37ºC) (Ware et al., 2005). Another study optimizing the same critical factors described an improved protocol consisting in: cooling the sample from 0 ºC to -35 ºC at a cooling rate of -0.5ºC/min, seeding at -10 ºC before being plunged immediately into the liquid nitrogen and rapid thawing. Under these conditions a survival rate of 80% was obtained (Yang et al., 2006). A successful usage of programmable freezing for the cryopreservation of adherent iPSCs has also been recently described (Katkov et al., 2011). The authors developed a six step programmed protocol including : 1) -1ºC/min from 0ºC (addition of CPA on ice) to -10ºC; 2) hold for 30 min at -10ºC; 3) -3ºC/min to -40ºC; 4) -1ºC/min to -60ºC; 5) -0.33ºC/min to -80ºC and 6) hold at -80ºC for 5 min and then transfer to liquid nitrogen. Adherent iPSC colonies cryopreserved using ethylene glycol as a CPA under these conditions showed a 63% recovery, which represents a 6 fold increase respect the preservation without a programmable freezer

Fig. 2. Effects occurring during the cryopreservation of cells at different cooling rates. When the cooling process starts, ice crystals formation is induced and free intracellular water is osmotically pulled from the cells. If the cooling process is slow this effect lead to cellular cell death by dehydration and shrinkage. In contrast, if the cooling process is rapid, intracellular ice crystals form before complete cellular dehydration has occurred. These crystals induce cell death by cellular organelles and membrane disruption during the thawing process. An optimal cooling rate together with the usage of cryoprotectants in the freezing media avoids dehydration effects and intracellular ice formation allowing cell survival after thawing.

Prevention of dehydration and shrinkage-induced cell death

**Optimal cooling + cryoprotectants** 

**Dehydration** 

**Rapid cooling** 

Cell death due to cellular dehydration and shrinkage

**Slow cooling** 

Cell death due to intracellular ice formation

using DMSO (Katkov et al., 2011).

**Ice crystals** 

**Non-permeating cryoprotectants Permeating cryoprotectants** 

**Cooling** 

Most of the studies carried out so far for the cryopreservation of dissociated hPSCs involved the usage of ROCK inhibitors (usually 10µM of Y-27632), since in its absence very few or none colonies are obtained. This inhibitor has been reported to significantly increase the survival rate of frozen/thawed single hESC as well as iPSCs (Claassen et al., 2009; Li et al., 2009; Martin-Ibanez et al., 2008; Mollamohammadi et al., 2009; Xu et al., 2010a; Xu et al., 2010b). Recent studies have demonstrated that Y-27632 increased not only the survival rate but also the adhesion of frozen–thawed dissociated single hPSCs in the presence and absence of feeder cells (Claassen et al., 2009; Katkov et al., 2011; Li et al., 2009; Martin-Ibanez et al., 2008; Mollamohammadi et al., 2009; Xu et al., 2010a; Xu et al., 2010b). In fact, Li et al. proposed that Y-27632 does not block apoptotic pathways, but rather prevents hPSCs from sensing their external environment, giving them time to make important cell-cell interactions and thus allowing them to escape anoikis (Krawetz et al., 2009; Li et al., 2009). Moreover, Mollamohammadi et al showed by RT-PCR analysis that the expression of integrin chains aV, a6 and b1 increased significantly in the presence of ROCK inhibitor (Mollamohammadi et al., 2009). They proposed that this increase in integrins expression may account for the maintenance of an undifferentiated state and an increase in cell adhesion of hESCs and iPSCs to the substrate allowing better cloning efficiency (Mollamohammadi et al., 2009).

The usage of ROCK inhibitors for continuous treatments has not induced any adverse effects on hPSCs pluripotency or chromosomal stability, even after substantial number of passages (Mollamohammadi et al., 2009; Watanabe et al., 2007). ROCK inhibitors such as Y-27632 or Fasudil are already used clinically in cardiovascular therapies (Hu & Lee, 2005), suggesting that they are safe for the treatment of hPSCs. Although the exact mechanism of action of ROCK inhibitors is, at the moment, unknown and a lot of cross talks between signaling pathways occur, the usage of this compound opens a new field of study to improve hPSCs cryopreservation and culture protocols.

#### **3.6 Controlled-rate cryopreservation**

The cooling rate is one of the cryobiological variables associated with damage during slowcooling (Figure 2). When the cooling process is rapid, intracellular ice crystals form before complete cellular dehydration has occurred. These ice crystals disrupt cellular organelles and membranes and lead to cell death during the recovery (thawing) process. On the other hand, when the cooling process is slow, free intracellular water is osmotically pulled from the cells resulting in complete cellular dehydration and shrinkage. This can also cause cellular death but there is little agreement on the mechanisms involved. However, when the cooling rate is slow enough to prevent intracellular ice formation, but fast enough to avoid serious dehydration effects, cells may be able to survive the freezing and thawing process. This survival zone or window is readily observed in many bacteria and other prokaryotes, but for most eukaryotic cells it is nonexistent or very difficult to find without using cryoprotectants. These agents have little effect on the damage caused by fast freezing (intracellular ice crystal formation), but rather prevent or lessen the damage caused by slow freezing (dehydration and shrinkage) (Figure 2) (Mazur, 1984). Thus, a tight control of the cooling rate is crucial to reduce cellular damage during cryopreservation, even in the presence of CPAs. This is achieved by the usage of programmable freezers. These devices, although expensive and not always available, are technically more reliable and reproducible. Several works have studied the relevance of programmable freezers for the cryopreservation of hPSCs. Ware et al reported survival rates of 60-70% with no apparent increase in differentiation using DMSO as a cryoprotectant, a control rate freezing device and straws as containers (Ware et al., 2005). The

Most of the studies carried out so far for the cryopreservation of dissociated hPSCs involved the usage of ROCK inhibitors (usually 10µM of Y-27632), since in its absence very few or none colonies are obtained. This inhibitor has been reported to significantly increase the survival rate of frozen/thawed single hESC as well as iPSCs (Claassen et al., 2009; Li et al., 2009; Martin-Ibanez et al., 2008; Mollamohammadi et al., 2009; Xu et al., 2010a; Xu et al., 2010b). Recent studies have demonstrated that Y-27632 increased not only the survival rate but also the adhesion of frozen–thawed dissociated single hPSCs in the presence and absence of feeder cells (Claassen et al., 2009; Katkov et al., 2011; Li et al., 2009; Martin-Ibanez et al., 2008; Mollamohammadi et al., 2009; Xu et al., 2010a; Xu et al., 2010b). In fact, Li et al. proposed that Y-27632 does not block apoptotic pathways, but rather prevents hPSCs from sensing their external environment, giving them time to make important cell-cell interactions and thus allowing them to escape anoikis (Krawetz et al., 2009; Li et al., 2009). Moreover, Mollamohammadi et al showed by RT-PCR analysis that the expression of integrin chains aV, a6 and b1 increased significantly in the presence of ROCK inhibitor (Mollamohammadi et al., 2009). They proposed that this increase in integrins expression may account for the maintenance of an undifferentiated state and an increase in cell adhesion of hESCs and iPSCs

to the substrate allowing better cloning efficiency (Mollamohammadi et al., 2009).

improve hPSCs cryopreservation and culture protocols.

**3.6 Controlled-rate cryopreservation** 

The usage of ROCK inhibitors for continuous treatments has not induced any adverse effects on hPSCs pluripotency or chromosomal stability, even after substantial number of passages (Mollamohammadi et al., 2009; Watanabe et al., 2007). ROCK inhibitors such as Y-27632 or Fasudil are already used clinically in cardiovascular therapies (Hu & Lee, 2005), suggesting that they are safe for the treatment of hPSCs. Although the exact mechanism of action of ROCK inhibitors is, at the moment, unknown and a lot of cross talks between signaling pathways occur, the usage of this compound opens a new field of study to

The cooling rate is one of the cryobiological variables associated with damage during slowcooling (Figure 2). When the cooling process is rapid, intracellular ice crystals form before complete cellular dehydration has occurred. These ice crystals disrupt cellular organelles and membranes and lead to cell death during the recovery (thawing) process. On the other hand, when the cooling process is slow, free intracellular water is osmotically pulled from the cells resulting in complete cellular dehydration and shrinkage. This can also cause cellular death but there is little agreement on the mechanisms involved. However, when the cooling rate is slow enough to prevent intracellular ice formation, but fast enough to avoid serious dehydration effects, cells may be able to survive the freezing and thawing process. This survival zone or window is readily observed in many bacteria and other prokaryotes, but for most eukaryotic cells it is nonexistent or very difficult to find without using cryoprotectants. These agents have little effect on the damage caused by fast freezing (intracellular ice crystal formation), but rather prevent or lessen the damage caused by slow freezing (dehydration and shrinkage) (Figure 2) (Mazur, 1984). Thus, a tight control of the cooling rate is crucial to reduce cellular damage during cryopreservation, even in the presence of CPAs. This is achieved by the usage of programmable freezers. These devices, although expensive and not always available, are technically more reliable and reproducible. Several works have studied the relevance of programmable freezers for the cryopreservation of hPSCs. Ware et al reported survival rates of 60-70% with no apparent increase in differentiation using DMSO as a cryoprotectant, a control rate freezing device and straws as containers (Ware et al., 2005). The

results of this study identified three critical factors for successful hESCs freezing: ice crystal seed at some point above the temperature of spontaneous intracellular ice formation (between -7ºC and -12ºC), an appropriate freezing rate (between -0,3ºC and -1,8ºC/min) and rapid thawing (at 25-37ºC) (Ware et al., 2005). Another study optimizing the same critical factors described an improved protocol consisting in: cooling the sample from 0 ºC to -35 ºC at a cooling rate of -0.5ºC/min, seeding at -10 ºC before being plunged immediately into the liquid nitrogen and rapid thawing. Under these conditions a survival rate of 80% was obtained (Yang et al., 2006). A successful usage of programmable freezing for the cryopreservation of adherent iPSCs has also been recently described (Katkov et al., 2011). The authors developed a six step programmed protocol including : 1) -1ºC/min from 0ºC (addition of CPA on ice) to -10ºC; 2) hold for 30 min at -10ºC; 3) -3ºC/min to -40ºC; 4) -1ºC/min to -60ºC; 5) -0.33ºC/min to -80ºC and 6) hold at -80ºC for 5 min and then transfer to liquid nitrogen. Adherent iPSC colonies cryopreserved using ethylene glycol as a CPA under these conditions showed a 63% recovery, which represents a 6 fold increase respect the preservation without a programmable freezer using DMSO (Katkov et al., 2011).

Fig. 2. Effects occurring during the cryopreservation of cells at different cooling rates. When the cooling process starts, ice crystals formation is induced and free intracellular water is osmotically pulled from the cells. If the cooling process is slow this effect lead to cellular cell death by dehydration and shrinkage. In contrast, if the cooling process is rapid, intracellular ice crystals form before complete cellular dehydration has occurred. These crystals induce cell death by cellular organelles and membrane disruption during the thawing process. An optimal cooling rate together with the usage of cryoprotectants in the freezing media avoids dehydration effects and intracellular ice formation allowing cell survival after thawing.

Cryopreservation of Human Pluripotent Stem Cells: Are We Going in the Right Direction? 157

advantage of being chemically defined, sterile and batch tested. The cryopreservation solution named STEM-CELLBANKERTM contains 10% DMSO, glucose and a high molecular weight polymer (undisclosed) used as a second cryoprotectant, all dissolved in phosphatebuffered saline. hPSCs are preserved using this solution in cryovials and the slow-cooling rapid-thawing method, without any programmed freezer. After thawing, cells are recovered in the washing solution named CELLOTIONTM containing NaCl, centrifuged to eliminate cryoprotectants and plated down on a feeder layer of human mitotically inactivated fibroblasts. Post-thaw recovery was substantially increased without any detrimental impact on proliferation or differentiation (Holm et al., 2010). Similar cryopreservation yields were obtained for both hESCs preserved as clumps and iPSCs preserved as single cells without ROCK inhibitor treatment. Therefore, this is a simple and efficient system that enables the cryopreservation of large quantities of hPSCs in a chemically defined medium that is clinical grade compatible (Holm et al., 2010). Employing a similar protocol but using a home-made cryopreservation solution containing 10% DMSO and 90% KSR, Li et al reported the preservation of single hESCs in serum and feeder-free conditions in the presence of ROCK

Understanding the mechanisms involved in the high vulnerability of hPSCs to the cryopreservation process is essential to develop efficient protocols for cryopreservation. Most of the research being undertaken over the last years is still empirical and few advances have been achieved in the identification of the pathways involved in the enhancement of cell survival induced by different factors, cryoprotectants or preservation systems. However, from the results obtained in these studies it is becoming increasingly clear that cell-cell adhesion and/or paracrine signaling between hPSCs are essential for survival and control of their undifferentiated state (Amit et al., 2000; Reubinoff et al., 2000; Thomson et al., 1998). Gap junctions and cell adhesion molecules are highly expressed in hESCs and have been implicated in these processes (De et al., 2002; Richards et al., 2004; Sathananthan et al., 2002; Wong et al., 2004; Wong et al., 2008). Therefore, disruption of these structures during cryopreservation due to ice crystal formation outside the cells may induce anoikis contributing to the poor recovery of hPSCs after slow cooling. However, a better understanding of this process together with a systematic study of the critical cryobiological variables is still needed to improve the already existing cryopreservation protocols. Further advances in the field would also require the development of reliable and standardized assays to measure not only immediate post-thaw recovery but also the ability of single cells or clumps to re-attach, proliferate and maintain pluripotency. Moreover, it is necessary to establish the n-points at which these assays should be applied, in order to allow direct quantitative comparisons between different cryopreservation methods that are not feasible at the moment. Thus, all present and future investigations would likely provide a reproducible effective and efficient cryopreservation protocol for hPSCs large-scale storage that will fulfill GMP requirements, permitting the widespread use of hPSCs in basic and/or

Our group is supported by grants from the Ministerio de Ciencia e Innovación (SAF2009- 07774 and PLE2009-0089), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación

inhibitor during the first day after thawing (Li et al., 2009).

**4. Conclusion** 

clinical research.

**5. Acknowledgements** 

A recent report made an interesting comparison of three methods of cryopreservation of hESC clumps including: conventional slow freezing-rapid thawing using cryovials, vitrification and programmable cryopreservation in plastic straws (Li et al., 2010b). Assessing the efficiency of cryopreservation by counting the number of attached undifferentiated colonies 1-2 days and 7-8 days after thawing they reached the conclusion that conventional cryopreservation may not be appropriated for hESCs preservation since few colonies attached and most of them were differentiated. The usage of a programmable freezer increased significantly the cryopreservation efficiency (~50% colony recovery respect to ~5% of conventional freezing), although it was not better than the high efficiency obtained by vitrification (80-90% colony recovery). Both methodologies maintain unaffected the pluripotency and normal karyotype of the cells (Li et al., 2010b). Another comparative study published at the same time reported lower survival rates after programmable cryopreservation of hESC clumps (10-20% survival colonies), although they were significantly higher than the ones obtained after conventional slow-freezing (4-8%) (Lee et al., 2010). In this study the best cryopreservation condition was obtained using a stepwise transfer method for hESC clumps, which consisted in using a series of solutions with increasing serum replacement and DMSO concentrations to achieve a stepwise equilibration before freezing. The same inverse process was performed after thawing in order to gradually rehydrate the cells. The combination of stepwise methods with programmable freezers yielded survival rates of 30-50% with low numbers of differentiated cells (Lee et al., 2010).

#### **3.7 Cryopreservation in xeno-free conditions**

Clinical application of hPSCs would need hESC and iPSC lines derived, cultured, differentiated and cryopreserved in xeno-free conditions following good manufacturing practice (GMP) regulations. Several attempts to improve hPSCs culture conditions have been reported. These advances include: the derivation of clinical grade hESC and iPSC lines, the use of conditioned media together with MatrigelTM as an attachment substrate for hPSCs culture and the derivation and propagation of hESC lines on human feeder layers in xenofree culture media (Amit et al., 2004; Hovatta et al., 2003; Rajala et al., 2007; Rajala et al., 2010; Richards et al., 2002; Richards et al., 2003; Skottman et al., 2006; Unger et al., 2008). Some approaches have also been done in the cryopreservation field towards the development of xeno-free effective cryopreservation protocols. The first one was an optimization of the established vitrification method previously described by Reubinoff et al (Reubinoff et al., 2001; Richards et al., 2004). In this new method they reported the successful vitrification of hESCs in sealed closed straws, their storage in the vapor phase of liquid nitrogen and the substitution of FCS with human serum albumin as the major protein source in the cryoprotectant solution. This refinement of the technique allowed the removal of animal components from the cryopreservation medium, therefore lowering the risk of cross-transfer of viruses and other pathogens to the hESCs. Moreover, sealing the straws the authors also prevented contact with potentially contaminated liquid nitrogen during cooling and storage. The efficiency of hESCs preservation was similar to the original vitrification protocol (Richards et al., 2004).

An effective serum and xeno-free chemically defined freezing procedure for hESCs and iPSCs has been recently developed (Holm et al., 2010). This protocol describes the usage of a commercially available freezing and post-thaw washing solution that presents the advantage of being chemically defined, sterile and batch tested. The cryopreservation solution named STEM-CELLBANKERTM contains 10% DMSO, glucose and a high molecular weight polymer (undisclosed) used as a second cryoprotectant, all dissolved in phosphatebuffered saline. hPSCs are preserved using this solution in cryovials and the slow-cooling rapid-thawing method, without any programmed freezer. After thawing, cells are recovered in the washing solution named CELLOTIONTM containing NaCl, centrifuged to eliminate cryoprotectants and plated down on a feeder layer of human mitotically inactivated fibroblasts. Post-thaw recovery was substantially increased without any detrimental impact on proliferation or differentiation (Holm et al., 2010). Similar cryopreservation yields were obtained for both hESCs preserved as clumps and iPSCs preserved as single cells without ROCK inhibitor treatment. Therefore, this is a simple and efficient system that enables the cryopreservation of large quantities of hPSCs in a chemically defined medium that is clinical grade compatible (Holm et al., 2010). Employing a similar protocol but using a home-made cryopreservation solution containing 10% DMSO and 90% KSR, Li et al reported the preservation of single hESCs in serum and feeder-free conditions in the presence of ROCK inhibitor during the first day after thawing (Li et al., 2009).
