临床研究用间充质干细胞的体外制备策略  

In vitro preparation of mesenchymal stem cells for clinical studies

作　　者：张文成[1] 王韫芳[1] 常铭洋[1] 池木根[2] 王华[2] 刘银霞[2] 覃金华[1] 闫舫[1] 裴雪涛[1] 吴祖泽[2] 

机构地区：[1]军事医学科学院野战输血研究所干细胞与再生医学实验室,北京100850 [2]军事医学科学院放射与辐射医学研究所,北京100850

出　　处：《中国医药生物技术》2012年第5期327-332,共6页Chinese Medicinal Biotechnology

摘　　要：目的探讨一种安全、有效并易于推广的临床研究用间充质干细胞的产业化制备策略。方法常规方法分离获得原代脐带间充质干细胞，P1-P3代以含小牛血清培养基进行稳定传代后，以无血清培养基对P4-P6代细胞继续培养和扩增，并以含血清培养基作为对照。倒置显微镜每天定时观察细胞生长状态，CCK-8法比较P4代和P6代细胞在两种培养基中的增殖能力，并对P4-P6代细胞的细胞裂解液和细胞培养上清中的牛血清白蛋白（BSA）残留量进行检测。结果对分离获得的原代脐带间充质干细胞进行传代培养，在含血清培养基中，P1-P3代细胞增殖能力稳定，以4×10^4／ml密度接种，3．5-4d可达到90％融合；细胞进入P6后，在无血清培养基培养条件下增殖能力与含血清培养基相近；BSA残留量检测结果显示，无血清培养基组中细胞的培养上清和细胞裂解液中残留的BSA量低于10ng／10^6细胞，满足《中国生物制品规程》对相关细胞治疗产品中BSA残留量的标准要求。结论含血清培养与无血清培养相结合的程序性培养扩增体系，可为临床提供数量充足、质量可控的间充质干细胞。Objective To establish a safe, effective and extendable strategy for in vitro preparation of mesenchymal stem cells （MSCs） for clinical studies. Methods UC-MSCs were mechanically isolated from umbilical cord of healthy newborns. After stably proliferating in the medium supplement with new born calf serum named SYL-NBCS through passagel（P1） to P3, cells were maintained in serum free medium named SYL-SF from P4 on with the cells cultured in the SYL-NBCS as control. Cell morphology of UC-MSCs cultured in different medium was observed by inverted microscope and the proliferation was analyzed with CCK-8 test. BSA residues were detected in both cell lysates and supernatant.Results UC-MSCs （P1-P3） reached 90% confluence after 3.5 - 4 days of culture in SYL-NBCS CCK8 analysis showed that UC-MSCs proliferation rate was similar when they were cultured in either SYL-SF or SYL-NBCS. Furthuremore, BSA residues testing demondtrated that the residuals of BSA in both cell lysate and culture supernatant cultured in SYL-SF were below 10 ng/10^6 cells, which met the safety value as Requirements of Biological Products. Conclusion The sequential culture of MSCs in serum-containing and serum-free culture medium provides a procedural amplification system for sufficient and quality controllable MSCs for either clinical studies or commercial applications.

关 键 词：间质干细胞 血清白蛋白 牛 生物疗 法 生物医学研究 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]