Attachment and growth of human embryonic stem cells on microcarriers
Attachment and growth of human embryonic stem cells on microcarriers
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DOI：
10.1016/j.jbiotec.2008.07.1997
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发表时间：
2008-11-06
影响因子：
4.1
通讯作者：
Crook, Jeremy Micah
中科院分区：
文献类型：
--
作者：
Phillips, Blaine Wesley;Horne, Rachel;Crook, Jeremy Micah
将人胚胎干细胞（hESC）用于基于细胞的疗法将需要大量遗传稳定的多能细胞及其分化的后代。传统的hESC增殖需要贴壁培养，并且对酶促解离敏感。这些限制阻碍了从二维平板培养中改进方法，这对于批量细胞生产来说是昂贵且不切实际的。用于临床的大规模培养将需要创新，例如用于生物加工的悬浮培养。在这里，我们描述了小鼠胚胎干细胞（mESCs）和人胚胎干细胞的附着和生长动力学的三甲基铵包被的聚苯乙烯微载体无饲养层，三维悬浮培养。mESC根据标准生长动力学粘附和扩增。对于hESC研究，我们测试了聚集体（胶原酶解离的）和单细胞（TrypLE（TM）解离的）培养。细胞迅速附着在珠子上，然后增殖。单细胞培养物在约5天内扩增3倍，略微超过hESC聚集体。重要的是，单细胞培养物保持6代，细胞数增加14倍，同时仍表达未分化标志物Oct-4和Tra 181。最后，hESC保留了它们向胰腺、神经元和心肌细胞谱系分化的能力。我们的研究提供了基于悬浮液的hESC在微载体上的扩增的原理证明，作为批量hESC生产的新颖、经济和实用的无饲养层的手段。(C)2008 Elsevier B. V.保留所有权利。
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 (TM)-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. (C) 2008 Elsevier B.V. All rights reserved.