Application of sugars and starch in the cryopreservatio of cells
Fibroblasts, keratinocytes and mesenchymal stem cells (MSC) are a useful tool in the field of regenerative medicines and tissue engineering due to their ability of being functional cell types of mesodermal lineage. Storing and banking of these cells can provide to every potential patient with a source of allogenic or autologous cells and tissues which are an important factor for cell replacement therapies and tissue production. The most effective way to preserve cells and tissues is by cryopreservation. It usually involves the use of compounds that can be cytotoxic (DMSO) or xenogeneous (serum). The perfect cryoprotectant agent is non-toxic, non-antigenic and provide with high viability rate. Given that, sugars and starchers which are often use as natural cryoprotectors can be used as a cryoprotectant agents minimizing the adverse effect due to the presence of DMSO and serum. Here I showed the development of a novel DMSO- and serum-free cryosolution for the cryopreservation of stem cells based on HES, sorbitol and dextran in Ringer Acetate. MSC diffentiation potential and viability were not affected after cryopreservation demonstrating that MSCs are still functional. Furthermore, several attemtps in the cryopreservation field to find the perfect cooling rate and method are still in process. Fully comparison of different freezing rates and methods were performed on this dissertation for MSC, fibroblasts and keratinocytes cryopreservation. The 3 cell types can be cryopreserved with slow cooling and vitrification. The use of controlled rate freezing machine which is expensive and time consuming is unnecessary. In addition, I clearly demonstrated that primary and cell line fibroblasts, being both the same cell type, differ in the cryopreservation efficiency in suspension and as monolyer, being this dissertation the first report to show that freezing protocols cannot be applicable for primary cells and cell lines.