**3. Improvement of existing cryopreservation protocols**

Many laboratories have been working over the last 10 years in the development of new cryoprerservation protocols for hPSCs. The main aim of the vast majority of these protocols has been the improvement of cell recovery including: an enhancement of cell survival and a reduction of cell differentiation. To this end different approaches have been adopted: development of new cryopreservation protocols such as vitrification, usage of different cryoprotective agents or molecules to improve survival, xeno-free cryopreservation media, cryopreservation of adherent hPSC colonies or single cells and/or utilization of devices to control changes in temperature. Although each of these works (summed up bellow), provide some improvements in hPSCs recovery after cryopreservation, not many of them have addressed the key question: how the changes introduced in the cryopreservation protocols contribute to the enhancement in cell recovery?

#### **3.1 Vitrification and optimizations of the technique**

One of the first attempts to overcome the low survival rates experienced by hPSCs after cryopreservation using the standard slow freezing-rapid thawing method was the adaptation of the vitrification protocol. This technique was developed for the cryopreservation of bovine ova and embryos (Vajta et al., 1998) and it was successfully adapted for hESCs freezing by Reubinoff and colleagues some years ago (Reubinoff et al., 2001). The protocol requires stepwise exposure of colony fragments to two vitrification solutions of increasing cryoprotectant concentrations. This exposure is sequential and brief (60 and 26 seconds respectively either at room temperature or at 37ºC). The common components of the vitrification medium are DMSO and ethylene glycol. The composition of the vehicle solution varies, with differences in sucrose concentration, the presence or absence of serum and the buffer used. Using mixtures of cryoprotectants helps to reduce the

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

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

Many laboratories have been working over the last 10 years in the development of new cryoprerservation protocols for hPSCs. The main aim of the vast majority of these protocols has been the improvement of cell recovery including: an enhancement of cell survival and a reduction of cell differentiation. To this end different approaches have been adopted: development of new cryopreservation protocols such as vitrification, usage of different cryoprotective agents or molecules to improve survival, xeno-free cryopreservation media, cryopreservation of adherent hPSC colonies or single cells and/or utilization of devices to control changes in temperature. Although each of these works (summed up bellow), provide some improvements in hPSCs recovery after cryopreservation, not many of them have addressed the key question: how the changes introduced in the cryopreservation

One of the first attempts to overcome the low survival rates experienced by hPSCs after cryopreservation using the standard slow freezing-rapid thawing method was the adaptation of the vitrification protocol. This technique was developed for the cryopreservation of bovine ova and embryos (Vajta et al., 1998) and it was successfully adapted for hESCs freezing by Reubinoff and colleagues some years ago (Reubinoff et al., 2001). The protocol requires stepwise exposure of colony fragments to two vitrification solutions of increasing cryoprotectant concentrations. This exposure is sequential and brief (60 and 26 seconds respectively either at room temperature or at 37ºC). The common components of the vitrification medium are DMSO and ethylene glycol. The composition of the vehicle solution varies, with differences in sucrose concentration, the presence or absence of serum and the buffer used. Using mixtures of cryoprotectants helps to reduce the

the substrate, resulting in a detachment induced apoptosis or anoikis.

**3. Improvement of existing cryopreservation protocols** 

protocols contribute to the enhancement in cell recovery?

**3.1 Vitrification and optimizations of the technique** 

(Wagh et al., 2011).

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

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

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 increasing the possibility of contamination and cell infection.

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

Cryopreservation of Human Pluripotent Stem Cells: Are We Going in the Right Direction? 147

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

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

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.,

teratoma formation (Wu et al., 2005).

than by the process of CPA addition/removal.

2005).

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