Isolation,purification and cultivation of rat muscle-derived stem cells / 中国组织工程研究

ABSTRACT

BACKGROUND:In vitro screening and amplification are important links to harvest muscle-derived stem cells that are satisfactory to clinical requirement.OBJECTIVE:To probe into the method of isolation,culture and purification of skeletal muscle-derived stem cells from adult rats in vitro.METHODS:The skeletal muscle was obtained sterilely following adult Sprague Dawley rats were anesthetized.Muscle-derived stem cells were harvested using enzyme digestion with Ⅺ collagenase,Dispese and trypsogen,and then purified by Percoll density gradient centrifugation and differential adhesion method.Growth curves were recorded and MTT colorimetric technique was used to describe the effects of various kinds of inoculum density on cell growth.Cells were identified by immunocytochemistry.RESULTS AND CONCLUSION:Primary muscle-derived stem cells were less in volume,lower adherence and well refraction,appearing as globular or fusiform or spindle and slowly multiplication.Following subculture,complete medium containing 20%serum was added.Cell number was greatest when cell density was 1×109/L,which was the optimal density.Cells at passages 1-4 grew well.Cells showed desmin(+),CD34(+),CD45(-)and Sca-1(+)by immunocytochemistry.Results verified that high-purity muscle-derived stem cells can be obtained in vitro and amplified successfully following primary culture.