Optimization for dissociation and culture of mesenchymal stem cells derived from umbilical cord Blood

Mesenchymal stem cells (MSCs) derived from umbilical cord blood (UCB) can not only support hematopoietic stem cells (HSCs) expansion in vitro as stromal cells, but also alleviate complications and accelerate the recovery of hematopoiesis during hematopoietic stem cell transplantation. The ratio of successful isolation and culture of UCB-MSCs, however is only about 20%similar to 30% to date. Many cell culture parameters contribute to this outcome and hence optimization of culture conditions is critical to increase the probability of success. In order to improve this ratio and optimize the culture method for UCB-MSCs, factorial design were applied to investigate the main influencing factors, including cell inoculate density (ID), combination and dose of cytokines, presence of serum and stromal cells or not. The experimental results indicated that ID was the most significant influencing factor for UCB-MSCs culture when P < 0.1. Higher ID leaded to higher probability of success and better growth for UCB-MSCs. Then were cytokines, which could Stimulate the growth of UCB-MSCs effectively. Based on the high cell ID, the optimized culture condition was determined with cytokines IL-3 (15 mu g/L) and GM-CSF (5 mu g/L) added in the traditional medium of MSCs. Then the probability of obtaining UCB-MSCs can be increased up to 90% from 30% with traditional culture method. Moreover, the characteristics of UCB-MSCs were tested by flow cytometric analysis and multi-lineage differentiation identification for osteoblast, chondrocyte and adipocyte. The results showed that the fibroblast-like cells expressed MSCS Surface markers of CD13, CD29, CD105, CD166 and CD44 positively and CD34, CD45 and HLA-DR negatively. Meanwhile the cells could differentiate into osteoblasts, chondrocytes and adipocytes similarly to MSCs derived from bone marrow. In conclusion, all efficient protocol have been developed to culture UCB-MSCs by adding cytokines IL-3 (15 mu g/L) and GM-CSF (5 mu g/L).