搅拌式生物反应器培养促进拟胚体的形成及其向心肌细胞分化

中国生物工程杂志

2009, Vol. 29

Issue (11): 1-6

研究报告

搅拌式生物反应器培养促进拟胚体的形成及其向心肌细胞分化

何文俊,叶玲玲,李世崇,刘红,王启伟,王海涛,谢靖,陈昭烈^**

军事医学科学院生物工程研究所北京 100071

Stirred Bioreactor Cultivation Enhances the Efficiency of Embryoid Bodies Formation and Differentiation into Cardiomyocytes

HE Wen-jun,YE Ling-ling,LI Shi-chong,LIU Hong,WANG Qi-wei,WANG Hai-tao,XIE Jing,CHEN Zhao-lie

Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China

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摘要：

目的：摸索搅拌式生物反应器培养小鼠胚胎干细胞（mESC）的最佳条件，建立一种批量制备拟胚体（EB）的方法。方法：研究mESC不同接种密度及生物反应器初始搅拌速度对EB形成的数量和质量的影响，以细菌培养皿中形成的EB为对照，用抗坏血酸诱导其向心肌细胞分化，比较两种培养体系对EB心肌细胞分化潜能的影响，通过免疫荧光染色及RTPCR对ESC来源的心肌细胞进行鉴定。结果：当mESC接种密度为1×105~3×105个/ml，搅拌速度设定为15~30r/min时，搅拌式生物反应器能高效制备出大量相对均一的EB，EB中几乎没有坏死细胞。与细菌培养皿制备的EB相比，生物反应器培养的EB向心肌细胞分化的效率更高，并表达心肌特异性基因。结论：搅拌式生物反应器培养促进EB的形成及其向心肌细胞分化，是一种更为理想的EB培养系统。

关键词： 生物反应器; 小鼠胚胎干细胞; 拟胚体; 心肌细胞; 分化

Abstract:

Objective: To determine the optimal condition for mouse embryonic stem cells (mESC) culture with stirred bioreactor, and to develop a method for mass production of embryoid bodies (EB). Methods: The different initial cell concentrations of mESC and the initial stirring speed of bioreactor were investigated to determine the optimal condition for EB formation. Induced by ascorbic acid, the differentiation of EBs formed in stirred bioreactor into cardiomyocytes was compared with EBs formed in Petri dish. Immunofluorescence staining and RTPCR were used to identify the cardiomyocytes derived from mESC. Results: The formation of a large number of uniform relatively EBs was achieved in stirred bioreactor when mESC were seeded initially with 1×105~3×105 cells/ml and stirring speed was set to 15~30r/min. Most of cells in the EBs formed in bioreactor were viable. EBs produced in bioreactor differentiated into cardiomyocytes more efficiently compared with EBs from Petri dish. The cardiac specific genes were expressed in ESCderived cardiomyocytes. Conclusions: Stirred bioreactor culture could enhance the efficiency of EB formation and differentiation into cardiomyocytes, which may be a more ideal culture system for EB formation.

Key words: Bioreactor Mouse embryonic stem cells Embryoid body Cardiomyocytes Differentiation

收稿日期: 2009-07-14 出版日期: 2009-12-07

ZTFLH:

Q813.1

基金资助:

军队十一五计划课题青年学者资助项目（06Q86）

通讯作者: 陈昭烈 E-mail: chenzl23@hotmail.com

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引用本文:

何文俊叶玲玲李世崇刘红王启伟王海涛谢靖陈昭烈. 搅拌式生物反应器培养促进拟胚体的形成及其向心肌细胞分化[J]. 中国生物工程杂志, 2009, 29(11): 1-6.

HE Wen-Dun, XIE Ling-Ling, LI Shi-Chong, LIU Gong, WANG Qi-Wei, WANG Hai-Chao, XIE Jing, CHEN Zhao-Lie. Stirred Bioreactor Cultivation Enhances the Efficiency of Embryoid Bodies Formation and Differentiation into Cardiomyocytes. China Biotechnology, 2009, 29(11): 1-6.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2009/V29/I11/1

［1］	Evans M J, Kaufman M H. Establishment in culture of pluripotential cells from mouse embryos. Nature, 1981, 292(5819):154~156
［2］	Dang S M, Kyba M, Perlingeiro R,et al. Efficiency of embryoid body formation and hematopoietic development from embryonic stem cells in different culture systems. Biotechnol Bioeng, 2002, 78(4):442~453
［3］	Cameron C M, Hu W S, Kaufman D S. Improved development of human embryonic stem cellderived embryoid bodies by stirred vessel cultivation. Biotechnol Bioeng, 2006, 94(5):938~948
［4］	Schroeder M, Niebruegge S, Werner A, et al. Differentiation and lineage selection of mouse embryonic stem cells in a stirred bench scale bioreactor with automated process control. Biotechnol Bioeng, 2005, 92(7):920~933
［5］	Fok E Y, Zandstra P W. Shearcontrolled singlestep mouse embryonic stem cell expansion and embryoid bodybased differentiation. Stem Cells, 2005, 23(9):1333~1342
［6］	GerechtNir S, Cohen S, ItskovitzEldor J. Bioreactor cultivation enhances the efficiency of human embryoid body (hEB) formation and differentiation. Biotechnol Bioeng,2004, 86(5):493~502
［7］	Lü S, Liu S, He W,et al. Bioreactor cultivation enhances NTEB formation and differentiation of NTES cells into cardiomyocytes. Cloning Stem Cells,2008, 10(3):363~370
［8］	Dang S M, GerechtNir S, Chen J,et al. Controlled, scalable embryonic stem cell differentiation culture. Stem Cells, 2004, 22(3):275~282
［9］	Sachinidis A, Fleischmann B K, Kolossov E,et al. Cardiac specific differentiation of mouse embryonic stem cells. Cardiovasc Res, 2003, 58(2):278~291
［10］	何文俊, 王晶波, 王常勇,等. 抗坏血酸体外诱导小鼠胚胎干细胞向心肌细胞分化. 解剖学杂志, 2007, 30(4):392~396 He W J, Wang J B, Wang C Y, et al. Chinese Journal of Anatomy, 2007, 30(4): 392~396
［11］	Sato H, Takahashi M, Ise H,et al. Collagen synthesis is required for ascorbic acidenhanced differentiation of mouse embryonic stem cells into cardiomyocytes. Biochem Biophys Res Commun, 2006, 342(1):107~112
［12］	zur Nieden N I, Cormier J T, Rancourt D E,et al. Embryonic stem cells remain highly pluripotent following long term expansion as aggregates in suspension bioreactors. J Biotechnol, 2007, 129(3):421~432
［13］	Shimada N, Sokunbi G, Moorman S J. Changes in gravitational force affect gene expression in developing organ systems at different developmental times. BMC Dev Biol, 2005, 5(1):10

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