Phillips Blaine Wesley, Horne Rachel, Lay Tan Suat, Rust William Lathrop, Teck Tan Thong, Crook Jeremy Micah
Institute of Medical Biology, 8A Biomedical Grove, #06-06, Singapore 138648, Singapore.
J Biotechnol. 2008 Nov 6;138(1-2):24-32. doi: 10.1016/j.jbiotec.2008.07.1997. Epub 2008 Aug 14.
The use of human embryonic stem cells (hESCs) for cell-based therapies will require large quantities of genetically stable pluripotent cells and their differentiated progeny. Traditional hESC propagation entails adherent culture and is sensitive to enzymatic dissociation. These constraints hamper modifying method from 2-dimensional flat-bed culture, which is expensive and impractical for bulk cell production. Large-scale culture for clinical use will require innovations such as suspension culture for bioprocessing. Here we describe the attachment and growth kinetics of both murine embryonic stem cells (mESCs) and hESCs on trimethyl ammonium-coated polystyrene microcarriers for feeder-free, 3-dimensional suspension culture. mESCs adhered and expanded according to standard growth kinetics. For hESC studies, we tested aggregate (collagenase-dissociated) and single-cell (TrypLE-dissociated) culture. Cells attached rapidly to beads followed by proliferation. Single-cell cultures expanded 3-fold over approximately 5 days, slightly exceeding that of hESC aggregates. Importantly, single-cell cultures were maintained through 6 passages with a 14-fold increase in cell number while still expressing the undifferentiated markers Oct-4 and Tra 1-81. Finally, hESCs retained their capacity to differentiate towards pancreatic, neuronal, and cardiomyocyte lineages. Our studies provide proof-of-principle of suspension-based expansion of hESCs on microcarriers, as a novel, economical and practical feeder-free means of bulk hESC production.
将人类胚胎干细胞（hESCs）用于基于细胞的治疗需要大量基因稳定的多能细胞及其分化后代。传统的hESC增殖需要贴壁培养，并且对酶解敏感。这些限制阻碍了从二维平板培养进行改进，而二维平板培养对于大规模细胞生产来说既昂贵又不切实际。临床应用的大规模培养将需要诸如用于生物加工的悬浮培养等创新方法。在这里，我们描述了小鼠胚胎干细胞（mESCs）和hESCs在三甲铵包被的聚苯乙烯微载体上的附着和生长动力学，用于无饲养层的三维悬浮培养。mESCs按照标准生长动力学附着并扩增。对于hESC研究，我们测试了聚集体（胶原酶解离）和单细胞（TrypLE解离）培养。细胞迅速附着在微珠上，随后增殖。单细胞培养物在大约5天内扩增了3倍，略超过hESC聚集体。重要的是，单细胞培养物传代6次，细胞数量增加了14倍，同时仍表达未分化标记物Oct-4和Tra 1-81。最后，hESCs保留了向胰腺、神经元和心肌细胞谱系分化的能力。我们的研究提供了hESCs在微载体上基于悬浮培养进行扩增的原理证明，这是一种新颖、经济且实用的无饲养层大规模生产hESCs的方法。
