改良原代培养方法提高人胎盘绒毛膜间充质干细胞的产量

Improving the production of human placental chorionic-derived mesenchymal stem cells by modified primary culture method

目的建立获得大量P0代人胎盘绒毛膜间充质干细胞（hpcMSCs）培养方法。方法从胎盘绒毛膜分离出hpcMSCs，经初次培养和再次培养7d后，将原培养瓶中培养基、未贴壁组织和冲洗生理盐水离心后分别进行再次培养和第3次培养。用倒置显微镜观察细胞形态，CCK-8测定细胞生长曲线，流式细胞术检测细胞表面标记，成脂、成骨诱导分化试剂盒鉴定细胞的分化潜能。结果获得的hpcMSCs均为成纤维细胞样贴壁细胞，每例胎盘获得（25．54±3．38）×10^个P0代细胞，初次培养、再次培养和第3次培养获得的细胞数分别为（11．73±2．09）×10^6、（11．12±1．42）×10^6和（2．69±0．71）×10^6，培养时间分别为（12．00±0．64）d、（8．87±0．63）d和（12．33±0．80）d。再次培养时间和获得细胞数同初次培养、第3次培养差异均有统计学意义（均P〈0．05），初次培养和第3次培养的时间差异无统计学意义（P〉0．05），但第3次培养有时间延迟的趋势。3次培养的每培养瓶获得细胞数分别为（1．12±0．15）×10^6、（2．10±0．16）×10^6和（1．04±0．16）×10^6，再次培养获得细胞数同初次培养、第3次培养差异均有统计学意义（均P〈0．05），初次培养和第3次培养获得细胞数差异无统计学意义（P〉0．05），但第3次培养有细胞数量减少的趋势。3次培养的细胞生长曲线和表面标记的表达率无差异，成骨、成脂诱导分化检测均呈阳性。结论一份绒毛膜标本，通过3次培养，获得的P0代hpcMSCs细胞数量比初次培养翻倍，本方法可为再生医学提供足够的种子细胞。

Objective To establish a cultivating method for obtaining a large number of PO generation human placental chorionic-derived mesenchymal stem cells （hpcMSCs）. Methods The hpcMSCs were isolated from human placental chorion. After primary culturing and cuhuring for seven days, the culture medium, the non-adherent tissue and the douching normal saline of the primary culture were centrifuged and re-cultured twice. Celt morphology was observed by an inverted microscope. CCK-8 was used to measure the cell growth curve. Flow cytometry was used to detect cell surface markers. Adipogenic and osteogenic differentiation kits were used to assess the cell differentiation potential. Results The obtained hpcMSCs were fibroblast-like adherent cells and （25.54±3.38）×10^6 cells were obtained per placenta. The total yield of the primary culture, secondary culture and tertiary culture were （11.73±2.09）×10^6, （11.12±1.42）×10^6 and （2.69±0.71）×10^6, respectively, and the incubation time were （12.00±0.64） d, （8.87±0.63） d and （12.33±0.80） d. There was significant differences in incubation time between the secondary culture and the primary culture as well as the tertiary culture （all P〈0.05）, and there was no significant difference between the primary culture and the tertiary culture. However, the incubation time of the tertiary culture had an increasing trend （P〉0.05）. The yield per culture flask of the primary culture, secondary culture and tertiary culture were （1.12±0.15）× 10^6, （2.10 ±0.16）× 10^6 and （1.04±0.16） × 10^6, respectively. There was significant differences in the yield per culture flask between the secondary culture and the primary culture as well as the tertiary culture （all P〈0.05）, and there was no significant difference between the primary culture and the tertiary culture. However, the yield per culture flask of the tertiary culture had a decreasing trend （P〉0.05）. There was no difference among the three cultures in the growth curve and the expression of surface markers, and the osteogenic and adipogenic differentiation were all positive. Conclusions The P0 generation hpcMSCs isolated from a choriocarcinoma sample can be doubled by the three cultures compared with the primary culture, which can provide plenty stem cell source for the regenerative medicine.