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-中国组织工程与临床康复2007年1期目次

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*★

吴国平，周燕，谭文松

Growth and metabolism of human dermal fibroblasts
in two dimensional and threedimensional culture systems*★

Abstract

AIM： To investigate the characteristics in growth and metabolism of human dermal fibroblasts (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 fibroblasts 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 supernatant, so as to observe the metabolism of cells.

RESULT: 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 cell 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 cells for tissue engineering. Based on the growth and glucose metabolism characteristics of human dermal fibroblasts cultured in three-dimensional system, it is necessary to improve mass delivery inside cell-scaffold composites during engineered tissue preparation in vitro.

State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China

Wu Guo-ping★, Studying for master’s degree, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China
wuguoping325@yahoo.com.cn

Correspondence to: Tan Wen-song, Doctor, Professor, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China
wstan@ecust.edu.cn

Supported by：the National Natural Science Foundation of China, No.20576036*

Received: 2006-09-21
Accepted: 2006-11-20

摘要
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24 h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。
关键词：生物医学工程；二维培养；三维培养；代谢

吴国平，周燕，谭文松.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[J].中国组织工程研究与临床康复，2007，11(1):74-77
[www.zglckf.com/zglckf/ejournal/upfiles/07-1/1k-74(ps).pdf]

华东理工大学生物反应器工程国家重点实验室，上海市 200237

吴国平★，男，1980年生，上海市人，汉族，华东理工大学在读硕士，主要从事人工皮肤组织的研究。
wuguoping325@yahoo.com.cn

通讯作者：谭文松，博士，教授，华东理工大学生物反应器工程国家重点实验室，上海市 200237
wstan@ecust.edu.cn

国家自然科学基金资助（20576036）*

中图分类号:R318.18 文献标识码:A
文章编号:1673-8225
(2007)01-00074-04

收稿日期：2006-09-21
修回日期：2006-11-20
（06-50-9-6964/Y·X）

课题背景：本课题为国家自然科学基金资助（20576036）。作为人体皮肤组织体外构建过程及其生物反应器的工程学问题研究的组成部分，通过本课题的研究，了解二维和三维培养系统中人皮肤成纤维细胞对葡萄糖营养成分的需求，为培养条件的设计和材料结构特性的改进提供指导，并为工程化组织体外构建过程系统模型的建立提供基础的生物学数据。

热点资讯：目前国内外针对开发在生物学和生理学方面更接近于天然皮肤的人工皮肤已有较多的研究，但作为改进培养工艺和设计适用于人工皮肤体外构建的组织工程反应器所必需的依据，对成纤维细胞在二维和三维系统中的生长代谢特性及其差异仍缺乏足够的了解。

同行评价：皮肤组织工程研究是近年来相关领域中倍受学者关注的热点课题。利用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为三维培养系统，对人皮肤成纤维细胞进行体外培养，并对成纤维细胞在不同培养系统中的代谢特征进行比较，是文章的特色。实验立题较新颖，有一定学术创新，结果对皮肤工程化构建有科学意义和临床应用前景。