Successful scale-up of human embryonic stem cell production in a stirred microcarrier culture system
Successful scale-up of human embryonic stem cell production in a stirred microcarrier culture system
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DOI：
10.1590/s0100-879x2009000600007
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发表时间：
2009-06-01
影响因子：
2.3
通讯作者：
Rehen, S.K.
中科院分区：
文献类型：
--
作者：
Fernandes, A.M.;Marinho, P.A.N.;Rehen, S.K.
人类胚胎干细胞（hES）的未来临床应用将需要高产量的培养方案。目前，hES细胞主要在静态组织板中培养，其提供有限的表面并且需要重复传代培养。在这里，我们描述了一个搅拌系统与商业葡聚糖为基础的微载体涂有变性胶原蛋白，以扩大人胚胎干细胞的生产。通过免疫细胞化学和流式细胞术分析多能性相关标志物，显示了基于微载体的搅拌系统中多能性的维持。形成表达内胚层、外胚层和中胚层标记物的空泡胚状体进一步证明了分化能力的维持。每体积培养基的细胞产量比静态平板高2倍以上，从而显著降低了培养成本。总共10（8）个核型稳定的hES细胞从单一的小容器中获得，其在细胞增殖期间几乎不需要操作，降低了污染的风险。转瓶的工作体积可达几升。如果需要，可以从均匀悬浮液中提取样品，以进行移植前最后扩增步骤所需的工艺验证。特别是在考虑涉及数十至数百名患者的临床试验时，使用少量较大的旋转器而不是数百个板或烧瓶将是有益的。据我们所知，这是第一次描述在连续搅拌下成功扩大未分化hES细胞的无饲养层和Matrigel（TM）生产，这使得该系统成为治疗和研究需求的有希望的替代方案。
Future clinical applications of human embryonic stem (hES) cells will require high-yield culture protocols. Currently, hES cells are mainly cultured in static tissue plates, which offer a limited surface and require repeated sub-culturing. Here we describe a stirred system with commercial dextran-based microcarriers coated with denatured collagen to scale-up hES cell production. Maintenance of pluripotency in the microcarrier-based stirred system was shown by immunocytochemical and flow cytometry analyses for pluripotency-associated markers. The formation of cavitated embryoid bodies expressing markers of endoderm, ectoderm and mesoderm was further evidence of maintenance of differentiation capability. Cell yield per volume of medium spent was more than 2-fold higher than in static plates, resulting in a significant decrease in cultivation costs. A total of 10(8) karyotypically stable hES cells were obtained from a unitary small vessel that needed virtually no manipulation during cell proliferation, decreasing risks of contamination. Spinner flasks are available up to working volumes in the range of several liters. If desired, samples from the homogenous suspension can be withdrawn to allow process validation needed in the last expansion steps prior to transplantation. Especially when thinking about clinical trials involving from dozens to hundreds of patients, the use of a small number of larger spinners instead of hundreds of plates or flasks will be beneficial. To our knowledge, this is the first description of successful scale-up of feeder-and Matrigel (TM)-free production of undifferentiated hES cells under continuous agitation, which makes this system a promising alternative for both therapy and research needs.