换液频率对骨髓间充质干细胞生物学特性的影响

Effects of medium exchange condition on biological characteristics of bone marrow mesenchymal stem cells

目的:培养条件是影响骨髓间充质干细胞生物学特性的重要因素。实验考察换液频率对兔骨髓间充质干细胞增殖分化及代谢特性等的影响。方法:实验于2005-03/2005-06在华东理工大学生物反应器工程国家重点实验室完成。①实验材料:1月龄新西兰大白兔购自上海市淞江车墩实验动物良种场。实验过程中对动物处置符合动物伦理学标准。②实验方法:采用密度梯度离心法从兔股骨骨髓中分离得到骨髓间充质干细胞,体外培养扩增后,取生长良好的第3代细胞分别以24,48,72h时间间隔进行换液培养。③实验评估:观察细胞形态学变化,测定细胞生长曲线同时进行乳酸和氨代谢分析,并对几种条件下收获的细胞进行集落形成和成骨分化检测。结果:①每24h换液的细胞最早进入对数生长期,第5天达到增殖顶点,最大细胞数目可达3.44×105,分别是48h和72h换液频率的1.43倍和1.71倍。②每48h换液条件下收获的细胞具有最强的集落形成能力,明显高于每24h和每72h换液条件下收获的细胞。③3种换液频率条件下收获的细胞经成骨诱导后茜素红染色均为阳性,其中每48h换液的细胞胞外钙基质分泌最高。④3种换液频率条件下细胞的代谢曲线无明显差异,乳酸和氨均维持较低浓度,分别在5mmol/L和2mmol/L以下。结论:①换液频率对骨髓间充质干细胞的影响具有双向性。提高换液频率促进骨髓间充质干细胞的增殖,同时也加速了干细胞特性的丢失,导致集落形成能力和成骨分化能力下降。②普遍采用的三四天换液不能提供适合骨髓间充质干细胞生长同时利于干细胞特性维持的营养环境,提示可通过常规培养条件的优化使其有利于骨髓间充质干细胞执行对称的细胞分裂。

AIM： Culture conditions affect biological characteristics of bone marrow mesenchymal stem cells （MSCs）. This research was aimed to investigate the effects of medium exchange condition on proliferation, differentiation and metabolism characteristics of MSCs. METHODS： The experiment was carried out in the State Key Laboratory of Bioreactor Engineering of East China University of Science and Technology from March to June 2005. （1）One-month-old New Zealand rabbits were bought from Chedun experimental animal farm in Shanghai Songjiang. During the experiments, the treatment of animals accorded with the animal ethics standards. （2）MSCs were isolated by the methods of density gradient centrifugation from bone marrow of New Zealand rabbit femur. After ex-vivo expansion, 3-generation MSCs were cultured under different medium exchange condition every 24, 48 and 72 hours. （3）The morphologic features, the growth curves and lactic acid and ammonia metabolic curves were observed. The colony forming efficiency and osteogenic differentiation potential of MSCs harvested under the different medium exchange conditions were determined. RESULTS： （1）MSCs under the medium exchange condition of every 24 hours entered exponential growth stage firstly and reached the peak on the 5^th day. 3.44×10^5 MSCs were obtained and the cell number was 1.43 and 1.71 times compared with the other two medium exchange conditions. （2）MSCs harvested under the medium exchange conditions of every 48 hours exhibited highest colony-forming efficiency compared with those under every 24 hours and 72 hours. （3）After osteogenic induction, MSCs harvested under three medium exchange conditions were all stained positive by Alizarin Red and those exchange medium every 48 hours produced highest calcium deposition. （4）The metabolism curves under three conditions had no significant differences. The lactic acid and ammonia concentrations kept in low level, less than 5 mmol/L and 2 mmol/L respectively. CONCLUSION： （1） Medium exchange frequency is ambidirectional dominance on MSCs. Increasing medium exchange frequency accelerated MSCs proliferation and loss of stem cell characteristics, while decreased the capacity of colony-forming and osteoblastic differentiation as well. （2）The commonly used medium exchange condition could not meet the needs of MSCs growth and stem cell maintenance, and this provides novel insights into optimizing culture parameters to form symmetrical MSCs division.