Cryopreservation of Rat Bone Marrow Derived Mesenchymal Stem Cells by Two Conventional and Open-pulled Straw Vitrification Methods

Objective: Mesenchymal stem cells (MSCs) are obtained from a variety of sources, mainly the bone marrow. These cells have a great potential for clinical research, however they cannot stay alive for long periods in culture. The aim of this study is to determine whether vitrification can be a useful freezing method for the 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 the vitrification method and open-pulled straw (OPS) vitrification and stored in liquid nitrogen with ethylene glycol ficoll (EFS) as a cryoprotectant for two months. The morphology and viability of thawed MSCs were evaluated by trypan blue staining. Furthermore, pre and post cryopreserved MSCs were induced to osteocyte and adipocyte with corresponding osteogenic and adipogenic medium. Results: After thawing, the viability rates were 81.33% ± 6.83 for the vitrification method and 80.83% ± 6.4 for OPS vitrification, while the values in the pre-vitrification control group were 88.16% ± 6.3 (Mean ± SD, n = 6). Post-cryopreserved cells from both the vitrification method and OPS vitrification also had a similar cellular morphology and colony-formation that was indistinguishable from non-vitrified fresh MSCs. In addition, the resuscitated cells cultured in induction medium showed osteogenesis. Mineral production and deposition was detectable by alizarine red S staining. Moreover, by applying an adipogenic differentiation condition, both pre and post cryopreserved cells differentiated into adipocyte and lipid vacuole accumulation that was stained by oil red O. Conclusion: Vitrification is a reliable and effective method for the cryopreservation of MSCs.