Yan Yuanwei, Song Liqing, Tsai Ang-Chen, Ma Teng, Li Yan
Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, 2525 Pottsdamer Street, Tallahassee, FL, 32310, USA.
Methods Mol Biol. 2016;1502:119-28. doi: 10.1007/7651_2015_310.
Conventional two-dimensional (2-D) culture systems cannot provide large numbers of human pluripotent stem cells (hPSCs) and their derivatives that are demanded for commercial and clinical applications in in vitro drug screening, disease modeling, and potentially cell therapy. The technologies that support three-dimensional (3-D) suspension culture, such as a stirred bioreactor, are generally considered as promising approaches to produce the required cells. Recently, suspension bioreactors have also been used to generate mini-brain-like structure from hPSCs for disease modeling, showing the important role of bioreactor in stem cell culture. This chapter describes a detailed culture protocol for neural commitment of hPSCs into neural progenitor cell (NPC) spheres using a spinner bioreactor. The basic steps to prepare hPSCs for bioreactor inoculation are illustrated from cell thawing to cell propagation. The method for generating NPCs from hPSCs in the spinner bioreactor along with the static control is then described. The protocol in this study can be applied to the generation of NPCs from hPSCs for further neural subtype specification, 3-D neural tissue development, or potential preclinical studies or clinical applications in neurological diseases.
传统的二维（2-D）培养系统无法提供体外药物筛选、疾病建模以及潜在的细胞治疗等商业和临床应用所需的大量人类多能干细胞（hPSC）及其衍生物。支持三维（3-D）悬浮培养的技术，如搅拌式生物反应器，通常被认为是生产所需细胞的有前景的方法。最近，悬浮生物反应器也被用于从hPSC生成类脑微型结构用于疾病建模，显示了生物反应器在干细胞培养中的重要作用。本章描述了使用转瓶生物反应器将hPSC神经定向分化为神经祖细胞（NPC）球的详细培养方案。从细胞解冻到细胞传代，阐述了为生物反应器接种准备hPSC的基本步骤。然后描述了在转瓶生物反应器中从hPSC生成NPC的方法以及静态对照。本研究中的方案可应用于从hPSC生成NPC，以进行进一步的神经亚型鉴定、3-D神经组织发育，或用于神经疾病的潜在临床前研究或临床应用。
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