**2. Sensitivity of hPSCs to cryopreservation**

The techniques employed for the cryopreservation of hPSCs which include slow freezingrapid thawing and vitrification, have been shown to be refractory for these cells that present

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 molecular-

> • Cell healthiness (good aspect, absence of differentiation and /or contamination) • Cell density before harvesting

*Critical variables*

concentration

• Selection of vehicle solutions

 medium at room temperature • Cooling rate (-1 to -10ºC/min) • Usage of programmable freezers

• Ice crystal formation • Storage bellow -130ºC

liquid nitrogen

• Osmotic injury

• Mild centrifugation • Diluting medium • Cell density

• Gentle harvesting using non-aggressive enzymes • Selection of appropriate cryoprotective agents and

• Temperature of cryopreservation medium addition • Exposure time of hPSCs to cryopreservation

• Avoidance of contamination by direct contact with

• Exposure time of hPSCs to cryopreservation

• Sealing and labeling of storage vessels

• Temperature and time of thawing

• Recrystalization of intracellular ice

• Feeder dependent or independent culture • Usage of molecules to improve survival

medium at room temperature

• Time and process (stepwise)

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

The techniques employed for the cryopreservation of hPSCs which include slow freezingrapid thawing and vitrification, have been shown to be refractory for these cells that present

good recovery rates.

**2. Sensitivity of hPSCs to cryopreservation** 

based apoptotic cell death processes (Baust et al., 2009).

*Cryopreservation steps*

Harvesting the cells: Single cells Colony clumps

Addition of cryoprotective agents within a freezing vehicle

> Cooling down cell suspension

Long/short term storage at cryogenic temperatures

Rapid thawing (37ºC)

Removal of cryoprotective agents

Plating the cells for culture expansion

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 cryopreservation?

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

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 of Human Pluripotent Stem Cells: Are We Going in the Right Direction? 145

intrinsic toxicity of each, and the method published by Reubinoff et al utilized 20% DMSO, 20% ethylene glycol and 0.5 mol/l sucrose (Reubinoff et al., 2001). However, no studies have been reported so far to determine the permeability of the cells (or colony fragments) to either cryoprotectant, or the intrinsic toxicity of these components to hESCs (Hunt, 2011).

Extremely rapid cooling rates are required to achieve vitrification using this two-component system. This is accomplished by direct immersion into liquid nitrogen of open-pulled straws containing small droplets (typically 1-20 µl) of vitrification solution within which the colony fragments (< 10) are held. Straws are then generally transferred to liquid nitrogen for long

The thawing process has to be as well, as rapid as possible to avoid ice crystallization. This is accomplished by direct immersion of the vitrified samples into pre-warmed culture medium containing sucrose, followed by stepwise elution of the cryopreotectants using sucrose as an osmotic buffer. An alternative method with direct exposure to growth medium without stepwise elution of the cryoprotectants has also been used with no

Vitrification has been adopted by many groups as the method of choice for hPSCs cryopreservation based on several comparative studies reporting recovery rates of undifferentiated colonies of more than 75% after vitrification compared to the 5-10% obtained after slow-cooling and rapid thawing (Li et al., 2010b; Reubinoff et al., 2001; Richards et al., 2004; Zhou et al., 2004). However, this technique presents some limitations: it is labor intensive and technically challenging, it is not suitable for large amounts of cells and the contact between the liquid nitrogen and the cells carries the risk of contaminations (Vajta & Nagy, 2006). Some attempts to improve these limitations have been done so far. One approximation was proposed by Heng et al for the cryopreservation of adherent hESCs colonies (Heng et al., 2005). They designed a culture plate made of detachable screw-cap culture wells resistant to storage at low temperatures in liquid nitrogen envisioned to develop automated systems for handling bulk quantities of cells (Heng et al., 2005). Alternatively, a method combining the large holding volume of slow-cooling rapid-thawing in cryotubs with the high efficiency of vitrification was described by Li et al (Li et al., 2008a). In this protocol hESCs clumps (>70um) were harvested after passage and transferred to a nylon cell strainer, exposed to vitrification solutions and vitrified by direct immersion in liquid nitrogen. Using this bulk vitrification method, 30 times more hESCs clumps (100-150) can be vitrified in a cell strainer compared to the open pulled straws. In addition, comparable results to those obtained for the classical vitrification method were reported for the recovery rate, the degree of differentiation and the maintenance of the pluripotency of the surviving cells (Li et al., 2008a). A refinement of this technique, using a cryovial fitted with stainless steel mesh, produced similar results (Li et al., 2010a). Although this method is easy and efficient to perform it still presents the limitation of direct contact of the cells with liquid nitrogen

In order to avoid direct contact of the vitrification solution with the liquid nitrogen several methods have been developed. Usage of embryo straws sealed in both ends with a commercial plastic bag heat sealer was reported by Richards et al (Richards et al., 2004). This

noticeable deleterious effects (Hunt & Timmons, 2007; Reubinoff et al., 2001).

increasing the possibility of contamination and cell infection.

term storage (Vajta et al., 1997; Vajta et al., 1998).

death despite an almost stable transcriptome. Supporting this theory the analysis of the microarray showed differences in the expression of genes involved in cell communication, cell growth and maintenance, cell death, cell differentiation and cell proliferation (Wagh et al., 2011). In agreement, Li et al showed that increased cellular adhesion induced by the Rho associated kinase (ROCK) inhibitor Y-27632 enhances the survival of single hESCs after thawing (Li et al., 2009). To demonstrate this, they treated cryopreserved hESCs simultaneously with Y-27632 and EGTA, a calcium chelant that disrupts cadherin activity and therefore cell adhesion. This double treatment significantly reduced the capacity of hESCs to form colonies and cell viability after thawing (Li et al., 2009). These results point to a high sensitivity of cryopreserved hPSCs to the loss of adherence between cells and/or to the substrate, resulting in a detachment induced apoptosis or anoikis.

According to the high differentiation rates experienced by hPSCs after cryopreservation, Wagh et al observed a down-regulation of pluripotency markers such as nanog, sox2 or klf4 (Wagh et al., 2011). In agreement with these results, it has been shown that the pluripotency marker Oct-4 was significantly decreased after culturing cryopreserved hESCs for several days (Katkov et al., 2006). In addition, the freeze-thaw stress increases the expression of several genes involved in the differentiation processes such as embryonic morphogenesis, neurogenesis, ossification, tissue morphogenesis, regeneration and vasculature development (Wagh et al., 2011).
