Isolation,culture,identification and labeling of bone marrow-derived mesenchymal stem cell of rabbit in vitro

Objective:To explore a method of isolation,purification and culture of bone marrow-derived mesenchymal stem cells(MSCs) of rabbits in vitro for the sake of further study.Methods:Bone marrow tissue was harvested from bilateral posterior iliac crest and tibias bone of young New Zealand white rabbit.MSCs were isolated and proliferated by the method of whole bone marrow culture.Growth curves of the 2nd,3rd,4th,5th and 6th generation MSCs were drawn by MTT method. CD34,CD44,CD45,CD105 antigens of MSCs were identified by flow cytometry.The purified MSCs were labelled with BrdU and immunohistochemistry was performed to calculate and evaluate the labelling rate.Results:Primary cultured MSCs started adhering to plates 12 hours after seeding and spindle-shaped MSCs were observed under microscope and growing at a rapid speed.Confluence of MSCs reached to 80%7～8 days after seeding.From the MSCs growth curve,which was S shape,it was known that MSCs grew in a rapid stage at the 4th-8th day after seeding,and MSCs of 3rd～5th generation possessed higher activity.CD34(-) ,CD45(-) ,CD44(+) and CD105(+) were detected by flow cytometry,which confirmed finally that the cultured cells were MSCs.The positive rate of MSCs labeled by BrdU was 85%～90%.Conclusion:In this study,simple operation,high efficiency,economy and practice were characteristic of this method,and MSCs were isolated and proliferated stably and rapidly.Cultured MSCs possessed high activity of growing and amplification and were appropriate for further research. Application of BrdU labelling for MSCs in vitro was safe and reliable.