Method of mesenchymal stem cells culture in vitro

Objective To evaluate the simple method for rat bone marrow mesenchymal stem cells(MSCs) culture and proliferation and generate into cardiomyocytes, providing potential therapeutic application for heart failure after myocardial infarction. Methods MSCs was isolated from adult rats using density gradient centrifugation and anchoring culure routinely. The other methods was operated as following: isolated thighbone and shankbone from a SD rat. The bone marrow cells were inoculated to tank farming in 2×105/ml density. Immortalized cells were obtained by frequent subculture for 3 era. After being co-cultured with 5-azacytidine, observe the morphologic change. Results The MSCs attached and grew as fibroblastic cells at 24 hours after initial planting. The hematopoietic stem cells that did not attach to the dish were washed from the culture with each medium changes. MSCs grew rapidly, developed into visible symmetric colonies at about 3 days and reached confluence at 7 to 10 days. While the cells were permitted to proliferate to confluence, MSCs were spindle-shaped morphology,small and arranged like pectinate.The MSCs did not differentiate spontaneously during culture expansion and maintained a normal MSCs biological features. After being co-culture with 5-azacytidine,some of MSCs became spindle-like cells,like cardiomyocytes-shaped. Conclusion MSCs can be cultured by the simplified Wakitani method. Cardiomyocytes can be generated from MSCs.It suggests that MSCs might be a new cell sources for stem cells transplantation in heart failure therapy.