骨髓间充质干细胞与胶原三维培养系统的建立

The establishment of three-dimension culture system for bone marrow mesenchymal stem cells and collagen

目的建立骨髓间充质干细胞(MSCs)与胶原的三维培养系统。方法 采用贴壁法对(5.8±3.4)×105个原代大鼠骨髓MSCs进行分离培养,通过传代次数的增加对其进行纯化扩增,然后以鼠尾胶原为成份构建胶原膜,用扫描电镜和噻唑蓝(MTT)比色法观察MSCs在胶原膜上的增殖状况。结果MSCs在体外扩增16代后共可获得(4.0±3.3)×1012个细胞,扩增(6.8±1.0)×106倍。扫描电镜结果显示,大鼠MSCs与鼠尾I型胶原有良好的亲和性;MTT结果显示,接种胶原膜后MSCs增殖特性与接种前相比,差异无显著性(P>0.05)。增殖曲线显示MSCs最适接种密度为2×104/cm2。结论大鼠MSCs接种后在胶原膜上增殖状况良好,此三维培养系统适于植入皮肤受损动物模型。

Objective To establish three-dimension culture system for bone marrow mesenchymal stem cells (MSCs) and collagen derived from rat tails. Methods MSCs were isolated and cultured according to their adhesive property, and purified by passages culture in vitro. After seeding collagen membrane, scanning electron microscope was used to observe the structure of the membrane and proliferation of MSCs and MTT was used to measure the proliferative activities of MSCs. Results MSCs could be expanded about (4.0±3.3)×1012 after 16 passages of culture. It has been increased by (6. 8±1.0) × 106 times. Scanning electronic microscopy showed that the rat MSCs had high affinity with the rat tails type I collagen. The different proliferative abilities between MSCs culture alone and MSCs seeded collagen membrane were also tested by MTT assay. There were no significant differences between the two groups and the optimal cell density for culturing rat MSCs seeded collagen membrane was 2 × 104/cm2. Conclusion MSCs seeded collagen membrane is fit for transplantation in animal models.