旋转发生器培养扩增人皮肤成纤维细胞

Culture and expansion of human dermal fibroblasts using rotary cell culture system

目的:研究旋转发生器扩增人皮肤成纤维细胞的可行性。方法:利用组织贴壁法从人皮肤中分离出人真皮成纤维细胞(hDFBs),使用DIO标记细胞后结合微载体在旋转发生器(RCCS)中培养,细胞贴附微载体的状况使用荧光显微镜、扫描电镜观测。并且检测细胞周期和分析细胞群体倍增时间,以比较微重力培养与平面培养的体外增殖能力差异。对照组细胞按常规平面培养。结果:在旋转发生器的微重力培养体系中,人成纤维细胞能快速贴附到微载体表面,在培养过程中达到很大的细胞密度,旋转发生器培养的细胞倍增时间为(2.99±0.50)d,对照组为(3.43±0.42)d(P<0.05);旋转发生器培养细胞增殖指数48.2±4.3,对照组35.2±3.4。结论:利用生物反应器和微载体悬浮培养人皮肤成纤维细胞,是大量制备皮肤组织工程种子细胞的一种有效方法。

Objective： To study the feasibility of culture and expansion of human dermal fibroblasts （hDFBs） on a large scale by Ro-tary Cell Culture System（RCCS）. Methods： hDFBs were cultured in vitro and labeled with fluorescent dye DIO, then the cells were transfered into RCCS containing culture medium and microcarrier Cytodex-3 at 5 g/L. The characteristics of fibroblasts on microearriers were observed with fluorescent microscope, and scanning electronic microscope （SEM）. Cell cycle and doubling time of RCCS cultured hDFBs were studied by cell counting and flow Cytometry and compared with that of conveitional plate cultured hDFBs （ con- trol）. Results： In RCCS culture hDFBs attached to Cytodex-3 and proliferated to heigh cell density. The doubling time（d） of RCCS cultured cells was 2.99± 0.5, that of controls 3.34 ± 0.42 （P 〈 O. 05 ）. The proliferation index of RCCS group was 48.2 ± 4.3, that of control 35.2 ± 3.4 （ P 〈 0.05 ）. Conclusion ： A culture system of biological reactor containing microcarriers is effective in preparation a large number of human skin fibroblasts.