**3.3 Addition of molecules to enhance survival**

One of the first molecules studied in the cryopreservation process to enhance cell survival was the caspase inhibitor Z-VAD-FMK (Heng et al., 2007). Results obtained in a previous work from the same group showing that apoptosis rather than necrosis was the responsible mechanism involved in the loss of viability during hESCs cryopreservation encouraged them to test a broad-spectrum irreversible inhibitor of caspase enzymes (Heng et al., 2006). Exposure to 100 mM Z-VAD-FMK in the freezing solution alone did not significantly enhace the postthaw survival rate. However, when Z-VAD-FMK was added to the freezing solution as well as to the post-thawing solution a significant enhancement in the cell survival rate (~two fold) was observed. Nevertheless, the differentiation rates of cryopreserved hESCs were not reduced and therefore the culture recovery was not improved (Heng et al., 2007). Similarly, the addition of a specific Caspase-9 inhibitor to the post-thawing recovering medium failed to increase hESCs colony formation 4-5 days after thawing, although it did reduce Caspase 8 and 9 activity 2 h after cryopreservation (Xu et al., 2010a). These results suggested that Caspase activity was not the triggering mechanism contributing to the low hPSCs recovery after cryopreservation, but it could be a downstream effector.

A significant improvement in the cryopreservation field came up with the addition of a ROCK inhibitor. ROCK have been found to play a role in the regulation of multiple biological pathways such as apoptosis, cell cycle, differentiation, cell adhesion as well as gene expression (Amano et al., 1997; Hall, 1994; Ishizaki et al., 1997; Krawetz et al., 2009; Maekawa et al., 1999). Watanabe et al reported for the first time, that addition of the ROCK inhibitor Y-27632 improved the cloning efficiency of dissociated hESCs more than 25-fold when the cells were plated at low density (Watanabe et al., 2007). One year later, the same inhibitor was tested for the cryopreservation of hESCs. Li et al demonstrated that 10 µM Y-27632 added to the post-thaw medium during 1 day increased hESCs survival when

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

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

One of the first molecules studied in the cryopreservation process to enhance cell survival was the caspase inhibitor Z-VAD-FMK (Heng et al., 2007). Results obtained in a previous work from the same group showing that apoptosis rather than necrosis was the responsible mechanism involved in the loss of viability during hESCs cryopreservation encouraged them to test a broad-spectrum irreversible inhibitor of caspase enzymes (Heng et al., 2006). Exposure to 100 mM Z-VAD-FMK in the freezing solution alone did not significantly enhace the postthaw survival rate. However, when Z-VAD-FMK was added to the freezing solution as well as to the post-thawing solution a significant enhancement in the cell survival rate (~two fold) was observed. Nevertheless, the differentiation rates of cryopreserved hESCs were not reduced and therefore the culture recovery was not improved (Heng et al., 2007). Similarly, the addition of a specific Caspase-9 inhibitor to the post-thawing recovering medium failed to increase hESCs colony formation 4-5 days after thawing, although it did reduce Caspase 8 and 9 activity 2 h after cryopreservation (Xu et al., 2010a). These results suggested that Caspase activity was not the triggering mechanism contributing to the low hPSCs recovery after

A significant improvement in the cryopreservation field came up with the addition of a ROCK inhibitor. ROCK have been found to play a role in the regulation of multiple biological pathways such as apoptosis, cell cycle, differentiation, cell adhesion as well as gene expression (Amano et al., 1997; Hall, 1994; Ishizaki et al., 1997; Krawetz et al., 2009; Maekawa et al., 1999). Watanabe et al reported for the first time, that addition of the ROCK inhibitor Y-27632 improved the cloning efficiency of dissociated hESCs more than 25-fold when the cells were plated at low density (Watanabe et al., 2007). One year later, the same inhibitor was tested for the cryopreservation of hESCs. Li et al demonstrated that 10 µM Y-27632 added to the post-thaw medium during 1 day increased hESCs survival when

protocol is suitable for handling bulk amounts of hPSCs (T'joen et al., 2011).

cryopreservation process (Ha et al., 2005).

**3.3 Addition of molecules to enhance survival** 

cryopreservation, but it could be a downstream effector.

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.


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 best condition tested in the work referenced.

Cryopreservation of Human Pluripotent Stem Cells: Are We Going in the Right Direction? 153

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.

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 the ROCK inhibitor treatments used for cryopreservation of hPSCs.

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

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