人皮肤成纤维细胞在二维和三维培养系统中的生长代谢特性

Growth and metabolism of human dermal fibroblasts in two-dimensional and three-dimensional culture systems

目的:观察人皮肤成纤维细胞在二维和三维培养系统中的生长和代谢特性。方法:实验于2005-02/2006-02在华东理工大学生物反应器工程国家重点实验室完成。从小儿包皮(患者家属知情同意并自愿捐献)真皮层中分离人皮肤成纤维细胞。分别以方瓶和微载体转瓶作为二维培养系统,以胶原-壳聚糖海绵状支架作为三维培养系统,将人皮肤成纤维细胞分别接种至不同系统中进行批培养和换液培养,每隔24h取样,采用结晶紫染色和MTT法检测细胞的增殖情况,用葡萄糖和乳酸检测试剂盒分析培养上清中葡萄糖和乳酸含量,以观察细胞的代谢情况。结果:对数生长期三维培养系统中人皮肤成纤维细胞平均比生长速率为二维培养系统中的0.25倍,葡萄糖比消耗速率为二维培养系统中的0.93倍,平均乳酸产率为二维培养系统中的1.56倍,细胞对葡萄糖的生长得率为二维培养系统中的0.26倍。比较人皮肤成纤维细胞在两种培养系统中增殖和代谢特性,发现三维培养的细胞比二维培养的细胞生长更缓慢,细胞中进行无氧代谢的葡萄糖占总葡萄糖代谢流量的比例高于二维培养的细胞,因此,葡萄糖代谢能量利用率低于二维培养的细胞。结论:二维培养系统,尤其微载体转瓶中,人皮肤成纤维细胞生长快速,葡萄糖代谢能量利用率高,适合于大量制备组织工程种子细胞。根据细胞在胶原-壳聚糖海绵状支架中生长代谢情况,要求工程化组织体外培养过程中应着重于改善三维支架内的物质传递情况。

AIM： To investigate the characteristics in growth and metabolism of human dermal flbroblasts （HDFs） in two-dimensional and three-dimensional culture. METHODS ： The experiment was carried out in the State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology between February 2005 and February 2006. HDFs were separated from children foreskin （admitted by their parents）. In current research, collagen-chitosan scaffolds were used as three-dimensional culture systems, T-flasks and microcarriers in spinner bottles were used as two-dimensional culture systems, in which human flbroblasts were cultured in vitro. The proliferation of samples was determined with crystal violet staining and MTT method, and glucose and lactic acid testing kits were adopted to analyze the contents of glucose and lactic acid in the supematant, so as to observe the metabolism of calls. RESULTS ： It was found that the specific growth rate in logarithmic phase, the specific glucose consumption rate, the average lactate yield on glucose consumption and the call yield on glucose consumption of human dermal fibroblasts on three-dimensional culture were 0.25 times, 0.93 times, 1.56 times and 0.26 times as high as those on two-dimensional culture, respectively. Compared with the growth and metabolism characteristics of HDFs on two-dimensional culture, cells grew slower and took a lower energy utilization rate on three-dimensional culture because more glucose metabolized through anaerobic pathway, which meant less ATP was yielded by per glucose molecule in cells. CONCLUSION：The above results indicate that two-dimensional systems, especially microcarrier-spinner culture system, are effective approaches to provide large amounts of seeding calls for tissue engineering. Based on the growth and glucose metabolism charactedstics of human dermal fibroblasts cultured in three-dimensional system, it is necessary to improve mass delivery inside call-scaffold composites during engineered tissue preparation in vitro.