Abrogation of E-cadherin-mediated cellular aggregation allows proliferation of pluripotent mouse embryonic stem cells in shake flask bioreactors.
Abrogation of E-cadherin-mediated cellular aggregation allows proliferation of pluripotent mouse embryonic stem cells in shake flask bioreactors.
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DOI：
10.1371/journal.pone.0012921
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发表时间：
2010-09-23
期刊：
影响因子：
3.7
通讯作者：
Ward CM
中科院分区：
文献类型：
--
作者：
Mohamet L;Lea ML;Ward CM
在再生医学中剥削胚胎茎（ES）细胞的基本要求是可重复地以成本效益的方式得出足够数量的一致质量的细胞，但是，由于培养的延伸性，不适合逐步培养的细胞构成，因此无法分化的ES细胞不适合悬浮培养。但是，悬浮的微载体或胚胎身体生物反应器培养物。
Here we show that acrogation of the cell surface protein E-cadherin, using either gene knockout (Ecad-/-) or the neutralizing antibody DECMA-1 (EcadAb), allows culture of mouse ES cells as a near-single cell suspension in scalable shake flask culture over prolonged periods without additional media supplements. Both Ecad-/- and EcadAb ES cells exposed adaptation phases in suspension culture, with optimal doubling times of 7.3 h±0.9 and 15.6 h±4.7分别在48小时内的可行细胞数量为95.1±2.0和16±0.9倍。
这是多能ES细胞的培养物作为手动喂食奶昔烧瓶生物反应器中的近节细胞悬浮液，代表了当前的ES细胞培养技术的显着改善，而这种原始方法将对人类ES和IPS细胞的培养物有用，以增加悬浮液的细胞膨胀率。
A fundamental requirement for the exploitation of embryonic stem (ES) cells in regenerative medicine is the ability to reproducibly derive sufficient numbers of cells of a consistent quality in a cost-effective manner. However, undifferentiated ES cells are not ideally suited to suspension culture due to the formation of cellular aggregates, ultimately limiting scalability. Significant advances have been made in recent years in the culture of ES cells, including automated adherent culture and suspension microcarrier or embryoid body bioreactor culture. However, each of these methods exhibits specific disadvantages, such as high cost, additional downstream processes or reduced cell doubling times. Here we show that abrogation of the cell surface protein E-cadherin, using either gene knockout (Ecad-/-) or the neutralising antibody DECMA-1 (EcadAb), allows culture of mouse ES cells as a near-single cell suspension in scalable shake flask culture over prolonged periods without additional media supplements. Both Ecad-/- and EcadAb ES cells exhibited adaptation phases in suspension culture, with optimal doubling times of 7.3 h±0.9 and 15.6 h±4.7 respectively and mean-fold increase in viable cell number of 95.1±2.0 and 16±0.9-fold over 48 h. EcadAb ES cells propagated as a dispersed cell suspension for 15 d maintained expression of pluripotent markers, exhibited a normal karyotype and high viability. Subsequent differentiation of EcadAb ES cells resulted in expression of transcripts and proteins associated with the three primary germ layers. This is the first demonstration of the culture of pluripotent ES cells as a near-single cell suspension in a manual fed-batch shake flask bioreactor and represents a significant improvement on current ES cell culture techniques. Whilst this proof-of-principle method would be useful for the culture of human ES and iPS cells, further steps are necessary to increase cell viability of hES cells in suspension.
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影响因子：
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