ISSN 0371-0874, CN 31-1352/Q



王秀丽, 王常勇, 虞星炬, 赵云山, 李晶, 段翠密, 郭希民

中国科学院大连化学物理研究所1802组. 辽宁, 大连 116023;军事医学科学院基础医学研究所组织工程研究中心. 北京 100850


以小鼠胚胎干细胞(ES-D3)为模型,应用新型细胞培养系统 ——STLV型旋转生物反应器(rotary cell culture system,RCCS)建立一种批量制备拟胚体(embryoid bodies, EBs)的新方法,研究不同细胞接种密度及培养时间对RCCS内EBs产生效 率的影响。为了进一步研究该制备方法是否对EBs的分化潜能产生影响,对照传统方法制备的EBs,利用形态学及RT- PCR方法测定经旋转生物反应器制备的EBs在自发性或诱导条件下(1% DMSO)向心肌细胞的分化能力。结果表明:ES-D3 在RCCS内能够高效形成EBs,与传统的直接悬浮法比较,其EBs的形成效率可达到后者的2倍。1 ×104个/ml为最佳细胞接种密度,培养时间也是在RCCS制备EBs过程中的重要因素之一,培养第4~5天为最佳收获EBs的时间。与悬滴法制备 的EBs比较,该方法制备的EBs分化为心肌细胞的潜能未改变。由此,应用旋转生物反应器可以高效制备EBs,该方法 制备的EBs可以用于发育生物学等基础及应用领域的相关研究。

关键词: 胚胎干细胞; 旋转生物反应器; 拟胚体; 微重力

Scalable production of embryoid bodies with the rotay cell culture system

Wang Xiuli, Wang Changyong, Yu Xingju, Zhao Yunshan, Li Jing, Duan Cuimi, Guo Ximin

Laboratory of Biomedical Material Engineering, Dalian Institute of Chemical and Physics, Chinese Academy of Science. Dalian 116023, China;Institute of Basic Medical Sciences, Academy of Military Medical Sciences. Beijing 100850, China


Embryonic stem (ES) cells are pluripotent cells capable of extensive proliferation while maintaining their potential to differentiate into any cell type in the body. ES cells can therefore be considered a renewable source of therapeutically useful cells. While ES-derived cells have tremendous potential in many experimental and therapeutic applications, the scope of their utility is dependent on the availability of relevant cell quantities. Therefore, most of the researches are being focused on the differentiation of ES cells. ES cell aggregation is important for embryoid body (EB) formation and the subsequent generation of ES cell derivatives. EB has been shown to recapitulate aspect of early embryogenesis, including the formation of a complex three-dimensional architecture wherein cell-cell and cell-matrix interactions are thought to support the development of the three embryonic germ layers and their derivatives. Standard methods of EB formation include hanging drop and liquid suspension culture. Both culture systems maintain a balance between allowing ES cell aggregation necessary for EB formation and preventing EB agglomeration for efficient cell growth and differentiation. However, they are limited in their production capacity. In this paper, we established a new approach for the mass production of EBs in a scalable culture system. The rotary cell culture system (RCCS, STLV type) was adopted to produce EBs. The vessel was placed on its rotary base and the experiment started with a beginning rotation rate of approximately 8 r/min which has been previously determined empirically as the optimal initial speed to yield randomized gravitational vectors while minimizing fluid shear stress. To keep the aggregations "floating in simulated microgravity" the rotation rate was increased as the EBs visibly grew. The EB production efficiency was calculated when different cell densities were inoculated. The kinetic change of EBs was measured during the time course of EB formation. Compared with the traditional method of producing EBs with hanging drop, the multi-potential of the resulting EBs in RCCS was analyzed by the capability of cardiomyocyte genesis. The results showed that EBs could be produced by RCCS with high efficiency. The optimal cell density inoculated in RCCS was 1×104 cells/ml, in which EB production was about twice higher than that in the suspending culture. Day 4~5 was the optimal time point for harvesting EBs. To clarify whether the differentiated potential of EBs might be affected by the microgravity produced by the rotary cell culture system, cardiogenic induction during ES cell differentiation was evaluated in our study. It was manifested by appearance of spontaneously and rhythmically contracting myocytes. In addition, immuno-histological and RT-PCR detection showed that the harvested EBs in RCCS exhibited the expected cardiac genesis and morphology. So, scalable production of EBs is obtained by RCCS. It will provide a useful approach to generate a large quantity of ES-derived cells for further research or application.

Key words: Embryonic stem cell;Rotary cell culture system;Embryoid bodies;Microgravity Received

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王秀丽, 王常勇, 虞星炬, 赵云山, 李晶, 段翠密, 郭希民. 应用旋转生物反应器批量制备拟胚体的研究[J]. 生理学报 2005; 57 (4): .

Wang Xiuli, Wang Changyong, Yu Xingju, Zhao Yunshan, Li Jing, Duan Cuimi, Guo Ximin. Scalable production of embryoid bodies with the rotay cell culture system . Acta Physiol Sin 2005; 57 (4): (in Chinese with English abstract).