**4. Conclusion**

156 Current Frontiers in Cryobiology

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.,

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

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

2010).

**3.7 Cryopreservation in xeno-free conditions** 

protocol (Richards et al., 2004).

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 clinical research.
