Uniform Embryoid Body Production and Enhanced Mesendoderm Differentiation with Murine Embryonic Stem Cells in a Rotary Suspension Bioreactor.
Embryonic stem cells (ESCs) are capable of differentiating into almost all cell types in vitro and hold great promise for drug screening, developmental studies and have a huge potential in many therapeutic areas. ESCs can aggregate to form embryoid body (EB) in static suspension culture by spontaneous differentiation, which resembles an intact embryo; while static suspension culture cannot prevent agglomeration of cells and offers little control over the size and shape of EBs, it results in aggregation of EBs into large, irregular masses, which prejudice the efficiency of differentiation of cells. Recently, bioreactor-based platforms have been shown to not only offer a beneficial effect on increasing diffusion of nutrients and oxygen which promotes cell viability and proliferation but also display local biomechanical properties (e.g., low fluid shear stresses and hydrodynamic force) in tissue development and organogenesis. This chapter describes a protocol for using a rotary suspension bioreactor to produce embryoid bodies and process the differentiation of mouse embryonic stem cells (mESCs), and to assess the efficiency of EB differentiation in the bioreactor by real-time PCR and immunostaining.
State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, Beijing, 100101, China.
Xiaohua
X
Lei
State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, Beijing, 100101, China.
Zhili
Z
Deng
State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, Beijing, 100101, China. duane@ioz.ac.cn.
Enkui
E
Duan
D000818:Animals; D019149:Bioreactors; D018929:Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods; D002454:Cell Differentiation; D002460:Cell Line; D002470:Cell Survival; D002478:Cells, Cultured; D058732:Embryoid Bodies / Q000166:cytology* / Q000378:metabolism; D004867:Equipment Design; D007150:Immunohistochemistry / Q000379:methods; D008648:Mesoderm / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods
Differentiation; EB formation; Mouse embryonic stem cell; Rotary cell culture system