王秀丽^1,2  王常勇^2,*  虞星炬¹  赵云山²  李 晶²  段翠密²  郭希民²

1中国科学院大连化学物理研究所1802组，辽宁 大连 116023；中国科学院研究生院

2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

摘 要: 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（RCCS）以建立一种批量制备拟胚体的新方法。明确接种不同细胞接种密度及培养时间对RCCS内拟胚体产生效率的影响。为了进一步明确该制备方法是否对拟胚体的分化潜能产生影响，对照传统方法制备的拟胚体，利用形态学及RT-PCR方法明确经旋转生物反应器制备的拟胚体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。10⁴/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天为最佳收获拟胚体的时间。与悬滴法制备的拟胚体比较，该方法制备的拟胚体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拟胚体,该方法的拟胚体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 Xiu-li ^1,2, WANG Chang-yong^2※, YU Xing-ju ¹, ZHAO Yun-shan², LI Jing²,

DUAN Cui-mi², Guo Xi-min²

1.Laboratory of Biomedical material Engineering, Dalian Institute of chemical and physics, Chinese Academy of Science, Dalian 116023, China.  (Graduate School of the Chinese Academy of Science)

2. Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.

Abstract: 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 ES cells’ differentiation. 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 EBs 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 rpm 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 EBs production efficiency was calculated when different cell densities were inoculated. The kinetic change of EBs was measured during the time course of EBs 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. Results showed that EBs could be produced by RCCS with high efficiency. The optimal cell density inoculated in RCCS was 10⁴ cells/ml, in which EBs production was about two times higher than that with the suspending culture method. 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 cells 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