种植密度、换液时间对人间充质干细胞原代培养时间的影响

Influence of the implantation density and medium change time on the primary culture time of human mesenchymal stem cells

目的 :探讨不同种植密度、换液时间对人间充质干细胞 (hMSCs)原代培养时间的影响 ,从而确定原代培养hMSCs的适宜条件。方法 :分离人骨髓细胞 ,按不同种植密度、首次换液时间分组进行原代培养 ,记录原代培养时间 ,然后进行传代培养和成骨诱导培养 ,并检测细胞碱性磷酸酶、骨钙素、钙结节表达情况。结果 :hMSCs原代培养种植细胞密度为 1× 10 5～ 3× 10 5/cm2 ,4 8h后换液的方法较合理。经化学药物 (如地塞米松、β -甘油磷酸、维生素C)成骨诱导后 ,hMSCs表达碱性磷酸酶、骨钙素明显增加 ,并可形成钙结节。结论 :合理hMSCs原代培养方法 ,可以快速获得大量的自体种子细胞 ,从而为组织工程化骨的体外构建创造条件。

Objective:To explore the influence of different implantation density and medium change time on the primary culture time of human mesenchymal stem cells(hMSCs)and ensure the optimum conditions for their primary culture.Methods:The isolated bone marrow cells were divided into groups according to different implantation density and first medium change time,and then were primarily cultured.The culture time was recorded.Then,secondary culture and osteogenesis-induced culture were performed on those cells.The expressions of alkaline phosphatase(AKP),osteocalcin and bone tubercula were all examined.Results:It was reasonable that hMSCs were plated at 1×10 5～3×10 5/cm 2cell density and the medium was changed after 48 hours.After osteogenesis induced by some chemicals(dexamethasone,a-glycerphosphoric sodium and vitamin C),the expressions of AKP and osteocalcin were significantly increased and bone tubercula were formed.Conclusion:Under optimal conditions,lots of autograft seeding cells can be obtained in short time and tissue engineering bone can be constructed in vitro.