Optimization of methods for in-vitro expansion and cryopreservation of mammalian stem cells.

摘要

The recent advances in the field of Tissue Engineering have given rise to the need for a tremendous quantity of cells. The search for and use of an appropriate multipotent or pluripotent stem cells in tissue engineering is an emerging concept.; Mesenchymal stem cells hold a very great deal of promise because of their capacity to self-renew and to differentiate into various lineages. These cells are very low in number in the body but their in vitro expansion capacity is very unique. Taking into consideration the lack of uniform approach for expansion of these MSCs and storing these cells for future use, in this work focus is laid on identifying the optimal culture and storage conditions for MSCs. The data obtained from this study indicates that the media and the quantity of the serum play a very important role in the expansion. Addition of HB-EGF has been shown to increase the proliferation.; The current viability rates, post recovery vary according to the cell type used. This gives rise to the need for analyzing various cells. In this thesis the experiments are based on two cell lines models. The viability rates obtained as the end result are used as a standard for optimizing the process for all mammalian cells. A pilot study involving rat mesenchymal stem cells was also performed to evaluate if the cryopreservation of stem cells is similar to the mammalian cell models. The results show that the viability rate of the cells post cryopreservation is high when there is 5% of DMSO in the freeze media and when the step-by-step slow cooling process is used. In conclusion, this thesis defines the optimal culture conditions for the expansion of MSCs in high numbers and storage of these cells for subsequent therapeutic approaches.