**1.2 Cryopreservation of hPSCs**

Cryopreservation is the process of cooling and storing cells, tissues or organs at sub-freezing temperatures, below −80°C and typically below −140°C, to maintain viability (Baust et al., 2009). The freezing process involves complex phenomena of water crystallization and changes in solute concentration both outside and inside the cell that can be detrimental to cell survival. In addition, exposure to low temperatures has been reported to induce a stress response resulting in biomolecular-based cell death for different cell types (Baust et al., 2001; Fu et al., 2001; Paasch et al., 2004; Xiao & Dooley, 2003).

In general, the major steps used in cryopreservation of most cell types can be summarized as follows (figure 1): i) harvesting the cells, ii) addition of cryoprotective agents within a carrier media to the cell suspension, iii) ice crystal induction in cell suspension following a determined cooling rate (ranging from -1 to -10ºC/min), iv) long-term storage at cryogenic temperatures (normally in liquid nitrogen), v) rapid thawing by warming the cell suspension in a 37-40ºC water bath, vi) removal of cryoprotective agent by centrifugation and vii) seeding down the cells to allow culture growth (Gao et al., 1998; Hubel, 1997).

Cryoinjury can be due to one or a combination of the following processes: 1) cytotoxicity of cryoprotective agents (Muldrew & McGann, 1994; Schneider & Maurer, 1983); 2) osmotic injury due to excursion of cryoprotective agents upon freeze-thawing (Gao et al., 1995; Mazur & Schneider, 1986); 3) intracellular ice formation in the cooling process (Fujikawa,

Cryopreservation of Human Pluripotent Stem Cells: Are We Going in the Right Direction? 143

very low survival rates (5-20% and 25-75% respectively) and many of the cells that do survive differentiate upon thawing and expansion (Reubinoff et al., 2001; Richards et al., 2004). The low efficiency of hPSCs cryopreservation has been attributed, in part, to the highly "cooperative" nature of these cells (as comparable with mESCs), which appear to require intimate physical contact between them within the colony (to permit cell-cell signaling) and an optimum clump size of about 100-500 cells during cryopreservation and serial passage (Amit et al., 2000; Reubinoff et al., 2000). All these statements mean that we are dealing with a cell type that presents extremely high sensitivity to cryopreservation. Therefore, the arising questions are: why are hPSCs so vulnerable to the cryopreservation process? And which are the processes involved in the low survival rates of hPSCs after

Heng et al postulated for the first time that apoptosis instead of cellular necrosis, was the major mechanism inducing the loss of viability of cryopreserved hESCs during freezethawing with conventional slow-cooling protocols (Heng et al., 2006). They showed that most of the cells were viable (~98%) immediately after thawing (determined by the Trypan blue dye exclusion method) and that cell viability was gradually decreasing with time in culture at 37ºC. Moreover, the kinetics of cell death could be reversibly slowed by a reduction in the temperature at which the cells were held post-thaw, indicating an apoptotic mechanism for cell death rather than an unregulated necrotic process. Based on these previous results, Xu et al investigated the apoptotic pathways activated during cryopreservation (Xu et al., 2010a). They described that the largest effect observed, mainly due to the freezing step, was an increase in the level of reactive oxygen species in hESCs. This presumably leads to the activation and translocation of p53 as strong expression of this protein was seen in the nucleus of thawed cells. Consequently, Caspase 9 was activated and a significant increase was also observed after thawing. In addition, Caspase 8 activity showed a similar increase post-thaw, indicating the possible activation of the extrinsic apoptotic pathway. They also stated that the elevated levels of F-actin observed during freezing could result in changes in apoptotic signals. These results led the authors to conclude that apoptosis in cryopreserved hESCs was induced through both, the intrinsic

However, a remaining question is unanswered: why hPSCs are so sensitive to apoptosis compared to mESCs or other cell lines? In order to answer this issue, Wagh et al performed detailed microarray studies on hESCs at different time points after thawing and compared their transcriptomes with control cells that did not go through the cryopreservation process (Wagh et al., 2011). Viability, stemness, colony morphology and proliferation were also monitored at different times post-thawing. They observed a full recovery of the phenotypes of cryopreserved hESCs after 5 days of cultivation. However, the number of colonies was significantly smaller in the frozen hESCs compared to control groups. Furthermore, the colony growth rate was also reduced. Gene expression analysis showed very similar transcriptomes for the surviving fraction of 30 minutes frozen-thawed hESCs and the control unfrozen cells. Therefore, they concluded that the transcriptome of the surviving hESCs is preserved during cryopreservation. On the other hand, increases in the number of the up- and down-regulated genes occur continuously within 24 h after thawing and culturing, and those genes are declined or maintained within 48 h. This observation favored the hypothesis that physical cellular damage induced by freezing and/or thawing inhibits proper attachment during cultivation resulting in an induction of anoikis apoptotic cell

cryopreservation?

and extrinsic pathways (Xu et al., 2010a).

1980; Mazur et al., 1972) and 4) recrystallization of the intracellular ice during the warming process (Mazur & Cole, 1989; Trump et al., 1965). In addition, recent studies have linked numerous stress factors associated with cryopreservation to known initiators of molecularbased apoptotic cell death processes (Baust et al., 2009).

Fig. 1. Representative diagram of the main steps involved in a general cryopreservation process and the critical variables that should be considered in order to preserve cells with good recovery rates.
