**2.2 Cryopreservation of MSCs**

4 Current Frontiers in Cryopreservation

freezing medium containing DMSO at 5-20%, transferred into glass or plastic cryovials and then frozen by cooling at 1.0 to 2.0 °C/min (28). Slow freezing reduces ice crystal formation and eliminates toxic and osmotic damage to cells through exposure to low concentrations of cryoprotectants while slowly decreasing temperatures (29). However, it is difficult to completely eliminate injury by intracellular ice formation. Damage by ice crystal formation in the cytoplasm during the freezing process is one of the possible causes of cell death; such conventional methods, are not applicable to hMSC cells because many of these cells die immediately after thawing (28). Alternately, vitrification, a rapid cooling method using a high concentration of cryoprotectant, could also be used. Vitrification can completely eliminate damage caused by ice crystal formation in the cytoplasm of cells during freezing (29, 30). It is also advantageous because the procedure takes a relatively short time and a

Vitrification has been used for the cryopreservation of oocytes, fertilized eggs and embryos of several mammalian species including humans in order to prevent ice crystal formation (32). There have been some reports demonstrating that embryonic stem (ES) cells could be successfully cryopreserved by vitrification in recent years (27, 33,34). Moon et al. tested vitrification of the human amnion-derived mesenchymal stem cells (HAMs) by using a twostep exposure to equilibration and vitrification solutions (21). They used an EG-based cryoprotectant and their findings were in line with previous reports that showed the superiority of EG. EG has been proven to be less toxic on fibroblast and other somatic cells in comparison with permeating agents such as DMSO and propylene glycol (PROH) that have been used on murine and human embryos (35). However, as a long-term preservation method for HAMs, a well-defined protocol of cryopreservation needs to be established for a human bone marrow derived mesenchymal stem cell bank. In the present study, to confirm the proliferative capability and pluridifferentiation of cryopreserved adult hMSCs; we chose ethylene ficoll sucrose (EFS) 40 that contained 40% v/v EG for the vitrification solution. hMSCs that were cryopreserved for two months were resuscitated and cultivated for 15 passages. An analysis of their expansion, morphological and pluridifferentiation characteristics was undertaken. Finally, under induction conditions, adipogenic and

For isolation of rat MSCs; female Sprague-Dawley rats (weighing 200-250 g) with the approval from the Institute for Animal Care were obtained from the Animal Center, Faculty of Medicine, Guilan University of Medical Sciences. Rats were killed by intraperitoneal administration of a lethal dose of sodium pentobarbital. The femurs and tibias were carefully dissected away from attached soft tissue as previously reported with modification (1). The ends of the bones were cut, and the bone marrow was aseptically extruded with 5 ml PBS solution by using a syringe with a 21G needle and flushing the shaft ten times. The marrow tissue was dissociated by pipetting. The cell suspension was then centrifuged at 500 × g for 5 minutes and the supernatant was discarded. Bone marrow mesenchymal stem cells (BMSCs) were then mechanically dispersed into a single-cell suspension so that the density of BMSCs reached 106 cells/ml. At this point, marrow cells were plated in a 25 cm² plastic flask in Dulbecco's modified eagle medium (DMEM) containing 20% fetal bovine serum

programmable temperature decreasing container is not required (31).

osteogenic potentials have been discussed.

**2.1 Preparation and culture of MSCs** 

**2. Materials and methods** 

MSCs at passage 4 of pre-cryopreservation were harvested and centrifuged at 400 × g for 15 minutes as mentioned above. Approximately 1 ×106 cells/ml of randomly selected batches were cryopreserved by using the vitrification method or OPS vitrification.
