Establishment of Feeder Layer-Free and Serum-Free Isolation and Culture System for Mouse Embryonic Stem Cells

摘要

The aim of this study was to establish a feeder layer-free and serum-free isolation and culture system for Kunming mouse Embryonic Stem Cells (ESCs). Mouse ESCs were isolated and cultured in Mouse Embryonic Fibroblasts (MEFs) feeder layer and Fetal Bovine Serum (FBS) culture system or feeder layer-free and serum-free culture system respectively and their primary colonies formation rates, maximum passage number and morphology and growth characters were compared. The undifferentiated status were identified by expression of Alkaline Phosphatase (AKP), Octamer-binding transcription factor 4 (Oct-4) and Stage-Specific Embryonic Antigen 1 (SSEA-1). The results indicated that there were no significant difference in the primary colonies formation rates (p<0.05) and no obvious difference in maximum passage number of ESCs between basal ESCs medium with MEFs feeder layer and FBS treated groups and modified ESCs medium without MEFs feeder layer and serum treated groups, respectively. There were some differences in morphology and growth characters of ESCs between those two kinds of culture systems. ESCs cultured in feeder layer-free and serum-free culture system were positive for AKP activity and the immunocytochemical staining studies revealed positive reaction to Anti-Oct-4 monoclonal antibody and Anti-SSEA-1 monoclonal antibody, respectively. Therefore, mouse ESCs isolated in this study can grow in feeder layer-free and serum-free culture system and maintain their self-renewal and undifferentiated state.