Isolation of murine muscle-derived stem cells with preplate technique combined with limited dilution technique

Background: Muscle-derived stem cells (MDSCs), a newly discovered adult stem cell possessing utility potential in tissue engineering and regenerative medicine, have been isolated from skeletal muscle tissue. The MDSCs isolating method varies a lot. Objective: To isolate and culture MDSCs and its clone as well as sub-clone through the use of modified preplate technique combined with limited dilution technique. Methods: The muscle tissue was obtained from new born mice under microscopy and then digested and filtered, from which MDSCs were isolated by modified preplate technique with first round plating period of 1 hour. The muscle derived cells were counted and MDSCs were marked with immunohistochemistry method. The MDSC clone and its subclone were obtained with limited dilution technique. Results and Conclusion: During the isolation procedure with preplate technique, muscle derived cells made up a progressively higher ratio in the cell culture and the procedure with first round plating period of 1 hour provided plenty cells for MDSCs isolation. MDSCs presented with adherent growth 72 hours after the sixth suspension, grew into cell population of 300-500 cells in 10 days about, and proliferated with its small round and spindle morphology persisted. MDSCs clone and sub-clone were obtained through limited dilution technique and found desmin positively expressed and Sca-1 positively expressed at ratio of (92.3±4.1)%. The MDSCs and its clone from mice may provide proper cell resource for tissue engineering and regenerative medicine research.