CAJ | 학술논문

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4×104/ml 密度接种,3.5~4 d 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10 ng/106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.
목적탐토일충안전、유효병역우추엄적림상연구용간충질간세포적산업화제비책략. 방법상규방법분리획득원대제대간충질간세포,P1~ P3대이함소우혈청배양기진행은정전대후,이무혈청배양기대P4~ P6대세포계속배양화확증,병이함혈청배양기작위대조.도치현미경매천정시관찰세포생장상태,CCK-8법비교 P4대화 P6대세포재량충배양기중적증식능력,병대P4~ P6대세포적세포렬해액화세포배양상청중적우혈청백단백(BSA)잔류량진행검측. 결과대분리획득적원대제대간충질간세포진행전대배양,재함혈청배양기중,P1~ P3대세포증식능력은정,이4×104/ml 밀도접충,3.5~4 d 가체도90%융합;세포진입 P6후,재무혈청배양기배양조건하증식능력여함혈청배양기상근;BSA 잔류량검측결과현시,무혈청배양기조중세포적배양상청화세포렬해액중잔류적 BSA 량저우10 ng/106세포,만족《중국생물제품규정》대상관세포치료산품중 BSA잔류량적표준요구. 결론함혈청배양여무혈청배양상결합적정서성배양확증체계,가위림상제공수량충족、질량가공적간충질간세포.
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 passage1(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/106 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.