应用旋转生物反应器批量制备拟胚体的研究  

Scalable production of embryoid bodies with the rotay cell culture system

作　　者：王秀丽[1] 王常勇[2] 虞星炬[1] 赵云山[3] 李晶[2] 段翠密[2] 郭希民[2] 

机构地区：[1]中国科学院大连化学物理研究所1802组 [2]军事医学科学院基础医学研究所组织工程研究中心 [3]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

出　　处：《生理学报》2005年第4期486-492,共7页Acta Physiologica Sinica

基　　金：Thisworkwassupportedby863ProgramofChinainTissueEngineering(No.2003AA205040);863ProgramofChinainSpaceTechnology(No.2004AA744052)andKeyProjectofBeijinginTissueEngineering(No.H020920050031).

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(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.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 tradition

关 键 词：胚胎干细胞 旋转生物反应器 拟胚体 微重力 

分 类 号：Q813.11[生物学—生物工程]