兔骨髓基质干细胞的分离生长及冻存技术研究

The isolation, growth and cryopreservation of rabbit marrow stromal cells

目的:建立兔骨髓基质干细胞(MSC)体外分离、培养、增殖、冻存的方法,以及探讨其生物学特性。方法:抽取兔髂骨及胫骨的骨髓液,采用密度梯度离心法结合贴壁培养法分离纯化出MSC,并增殖。观察MSC的生长特性,测定其贴壁率及第3、8、13、18代的生长曲线,并评价其生物学特性。MSC在12.5%DMSO条件下经液氮或-60℃冻存复苏,测其成活率。结果:MSC为贴壁生长,以均一的梭形的成纤维细胞样生长,贴壁及增殖能力强,所测各代的生长曲线呈相似的S型,倍增时间约为35h。MSC需液氮保存,短期也可-60℃冰箱保存。结论:获得的兔MSC生长旺盛、增殖能力强,其分离、培养、纯化、增殖、冻存方法是可行的,可较长时间稳定地培养增殖、冻存,传代的MSC可保持其原代的生物学特性。MSC将是组织工程理想的种子细胞。

Objective:To explore the optimal methods of isolating, culturing, puring, proliferating, and freezing of rabbit marrow stromal cells(MSC) in vitro, and investigate their biological properties.Methods: MSC were aspirated from rabbit iliac and tibias crest and purified by gradient centrifuge and adhesive culture methods.MSC were cultured and expanded in flasks.Growth curve and adhesive rate of passaged MSC were determined, and their biological characteristics were investigated.MSC were stored in liquid nitrogen or -60℃ refrigerator long term, and their survivability was determined.Results: The MSC in culture were uniform spindle-shaped in appearance and showed active proliferative capacity and had similar S shape of growth curves.The population double time of the subcultured MSC was about 35 h.MSC could be stored in liquid nitrogen long term.Conclusion: The methods of isolating, culturing, puring, expanding, and freezing of rabbit MSC are feasible.The MSC can be cultured, expanded and cryopreserved stably in vitro.The subcultured MSC can maintain their original biological properties.MSC can be optimal seed cells source for tissue engineering.