Successful Vitrification of Rat Bone Marrow Derived Mesenchymal Stem Cells (Pages: 0-0)


Bahadori M.H *, Soltani B , Mirzajani E , Babaee P , Ansar M.M ,

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Objective: Mesenchymal stem cells (MSCs) are obtained from a variety of sources, mainly bone marrow. These cells have great likely for clinical research due to their potential to regenerate tissue. A cryopreservation procedure for MSCs is required because these cells cannot stay alive for long periods in culture. The aim of this study was to determine whether vitrification is a useful freezing method for storage of MSCs. Materials and Methods: Mesenchymal stem cells were isolated from rat bone marrow based on their capacity to adhere to plastic culture surfaces. MSCs were cryopreserved using both vitrification method and OPS vitrification and stored at -196 °C in liquid nitrogen with EFS as cryoprotectant for two months. The morphology and viability of thawed MSCs were evaluated by Trypan Blue staining. Furthermore, pre and post cryopreserved MSCs induced to osteocyte and adipocyte with corresponding osteogenic and adipogenic medium for three weeks and alizarine red S and oil red- O staining were done. Results: We were able to obtain homogeneous plastic adherent cells from the mononuclear cell fractions of bone marrow using our culture conditions. After thawing, the viability rates was 81.33% ± 6.83 for vitrification method and 80.83% ± 6.4 for OPS vitrification, while the values with the before vitrification control group were 88.16% ± 6.3 (Mean ± SD, n = 6). Post-cryopreserved cells from both of vitrification method and OPS vitrification also had similar cellular morphology and colony-formation indistinguishable from the non-vitrified fresh MSCs. In addition the resuscitated cells cultured in induction medium consisting of 100 nM dexamethasone, 10 mM ß-glycerol phosphate and 50 µM ascorbic acid-2-phosphate, showed osteogenesis, and mineral production and deposition was detectable after 21 days by alizarine red S staining. Moreover, applying adipogenic differentiation condition, pre and post cryopreserved cells differentiated into adipocyte by 5µg/ml insulin, 1 µM dexamethasone, 100 nM Indomethacine, 0.5 mM methylisobutylxanthine and lipid vacuole accumulation was stained by oil red O. Conclusion: This study indicates that vitrification is a reliable and effective method for cryopreservation of MSCs.