**8. Conclusion**

12 Current Frontiers in Cryopreservation

survival rate compared with vitrification, indicating a high efficiency of the vitrification

Moreover, Heng (48) demonstrated that 39.8 ± 0.9% of the hMSCs could be recovered after cryopreservation using a conventional slow freezing method which was lower than that our result (Table 1). Here, resuscitated MSCs kept a high proliferative potential. They first grew as clones after limiting dilution and then expanded rapidly with the typical features of spindle-shaped cell bodies and confluence after a lag phase of 6-14 days. There were no differences among the pre and post-cryopreservation of colony formation at the same passage (Fig 2). In addition, the results showed that the passage procedure was selective for MSCs and it could be inferred that the passaging of resuscitated MSCs increased cellular

Ji et al. demonstrated that cryopreservation of encapsulated HES cells offers better cellular viability, higher colony recovery, and less differentiation than the slow-freezing techniques most commonly used to preserve HES colonies. Therefore, this difference in recovery may be due to differences in cell lines, freezing and thawing protocols, or growth substrate (49). On the other hand, previous studies have shown that cyropreservation had no effect on either the proliferation or osteogenic and adipogenic differentiation of human MSCs *in vitro*  (5, 22). In agreement with these reports, Liu et al. using slow cooling with Me2SO as a cryoprotectant and rapid thawing demonstrated that thawed cryopreserved human MSCs had higher survival rates in comparison with non-cryopreserved MSCs and differentiated into osteoblasts when cultured in osteogenic media. Also they found that cryopreserved hMSCs could not differentiate into osteoblasts spontaneously when cultured in basic culture media (50). In addition to the characteristics described above, our present study demonstrated that post-cryopreserved MSCs from bone marrow were still pluripotential and differentiated into osteoblasts and adipocyte under appropriate culture conditions (Fig 3, 4). These observations suggest that the "memory" of proliferation and differentiation in MSCs is not affected by the process of vitrification. The ability of frozen BM-MSCs by slow freezing to differentiate into mesenchymal derivatives (such as osteogenic and adipogenic) reported by Lee et al. (4). In this study, we established a two-step vitrification protocol for MSCs using EFS containing 40% v/v EG for the vitrification solution, which is widely used for successful vitrification of mouse embryos (30), human blastocysts (32) and ESCs (34). Our findings are in line with the reports by Gajda et al. who used the same methods for somatic cells which have been proven to be less toxic on bovine skin fibroblast and cumulus

EG is the most commonly used cryoprotectant for vitrification due to its low molecular weight and low toxicity (35). In addition, additives with high molecular weights, such as sucrose, can significantly reduce toxicity by decreasing the concentration of permeating agents required for the vitrification solution. We also used ficoll as a macromolecule to promote permeation by cryoprotectants, which seems to have the advantages of lower toxicity, higher solubility and lower viscosity (30). In a study by Moon et al. on HAMs, they observed that the combination of EG with either PROH or DMSO resulted in a very low survival rate of HAMs as compared with EFS alone (21). Also, Kuleshova and Lopata ascertained the advantages of vitrification when compared with earlier applied cryopreservation techniques (51). These advantages include the control of solute penetration

procedure (21).

homogeneity.

cells (35).

In the present experiment, it was shown that vitrification can be an efficient storage method for MSCs without losing their activity and usual properties. Such a system will be exceedingly helpful for both experimental research and medical applications.
