**1. Introduction**

The first derivation of human embryonic stem cells (hESCs) (Thomson et al., 1998) and the more recently development of human induced pluripotent stem cells (iPSCs) (Park et al., 2008; Takahashi et al., 2007; Takahashi & Yamanaka, 2006; Wernig et al., 2007; Yu et al., 2007) have marked the beginning of a new era in biomedical research. These two types of human pluripotent stem cells (hPSCs) are characterized by an unlimited capacity to selfrenew while retaining their potential to differentiate into almost all cell types of the body (Odorico et al., 2001; Reubinoff et al., 2000; Silva & Smith, 2008). These remarkable properties turn hPSCs into one of the most interesting cell types for toxicology and drug discovery, tissue engineering and regenerative medicine (Battey, 2007; Mountford, 2008). In fact, work with hPSCs has already provided new and exciting developments that may eventually lead to the creation of novel cell-based therapies for the treatment of a wide range of human diseases including Parkinson's and other neurodegenerative diseases, diabetes, cardiac and vascular diseases (Kiskinis & Eggan, 2010; Ronaghi et al., 2010). However, a major challenge for the widespread application of hPSCs is the development of efficient protocols for cryopreservation.

To date, two techniques are mainly applied for the cryopreservation of hPSCs: conventional slow freezing and vitrification. The conventional slow-freezing and rapid-thawing procedure using dimethylsulfoxide (DMSO) as a cryoprotectant is the most commonly used method (Grout et al., 1990; Meryman, 2007). While this established technique is effective for somatic cell lines and even murine embryonic stem cells (mESCs), hematopoietic and mesenchymal human stem cells, this is not the case for hPSCs, due to low recovery rates and high levels of differentiation (Berz et al., 2007; Reubinoff et al., 2001; Richards et al., 2004; Thirumala et al., 2010). In contrast, vitrification of hPSCs by the "open pulled straw" method

Cryopreservation of Human Pluripotent Stem Cells: Are We Going in the Right Direction? 141

An important revolution in the stem cell research field was accomplished when several groups in different studies demonstrated that using a cocktail of four factors, somatic cells could be reprogrammed into iPSCs (Maherali et al., 2007; Okita et al., 2007; Park et al., 2008; Takahashi et al., 2007; Takahashi & Yamanaka, 2006; Wernig et al., 2007; Yu et al., 2007). Consequently, it was shown that such cells could be generated from patient-specific cells for a wide variety of diseases (Kiskinis & Eggan, 2010; Raya et al., 2009; Raya et al., 2010) and from a wide variety of somatic cell types (Sun et al., 2010). These cells are morphologically similar to hESCs, express typical hESC-specific cell surface antigens and genes, differentiate into multiple lineages *in vitro*, and form teratomas containing differentiated derivatives of all three primary germ layers when injected into immunocompromised mice. Indeed, these new pluripotent cell lines satisfy all the original criteria proposed for hESCs (Thomson et al., 1998). Nevertheless, some differences have been observed between hESCs and iPSCs (Chin et al., 2009); but it remains unclear whether the small percentage of genes that are differentially expressed between these two types of hPSCs are shared among different lines

Developing iPSCs into therapeutic reagents faces a number of practical hurdles, including risks associated with cell processing, the difficulty of ensuring the purity and characteristics of the reprogrammed population and the safety and efficacy of reprogrammed cells *in vivo* (Condic & Rao, 2008; Rao & Condic, 2008; Rao & Condic, 2009). Moreover, a case of rejection has been recently described after iPSCs autologous transplantation (Apostolou & Hochedlinger, 2011). Nonetheless, there is cause for considerable optimism that patientspecific iPSC lines will both enhance the study of human diseases and advance these studies

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;

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,

**1.1.2 Human induced pluripotent stem cells** 

and whether these differences are biologically significant.

Fu et al., 2001; Paasch et al., 2004; Xiao & Dooley, 2003).

toward clinical applications.

**1.2 Cryopreservation of hPSCs** 

using high cryoprotectant concentrations together with flash-freezing in liquid nitrogen has reported higher cell survival rates (Li et al., 2010b; Reubinoff et al., 2001; Richards et al., 2004). However, there are several disadvantages preventing the widespread use of this technique. First, high concentrations of cryoprotectors, which are cytotoxic above 4ºC, are needed. Second, these procedures are tedious to perform manually. Additionally, as vitrification is mostly performed in open pulled straws, contact between the liquid nitrogen and the cells is unavoidable, which carries the risk of contamination. Finally, and one of the most limiting disadvantages of this technique is that it is clearly unsuited for freezing bulk quantities of hPSCs.

During the last decade, several groups have been studying different approaches to improve the above described cryopreservation protocols. In the present work we will review the recent advances in the cryopreservation field trying to point out how a better understanding of the sensitivity of hPSCs to the cryopreservation process will help to develop more efficient protocols.
