Cardiac differentiation of human pluripotent stem cells in scalable suspension culture
Cardiac differentiation of human pluripotent stem cells in scalable suspension culture
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DOI：
10.1038/nprot.2015.089
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发表时间：
2015-09-01
期刊：
影响因子：
14.8
通讯作者：
Zweigerdt, Robert
中科院分区：
文献类型：
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作者：
Kempf, Henning;Kropp, Christina;Zweigerdt, Robert
由人多能干细胞（hPSC）产生的心肌细胞（CM）是用于再生疗法、药物发现和疾病建模的潜在细胞来源。所有这些应用都需要常规供应相对大量的体外生成的CM。该方案描述了用于将hPSC-CM生成为仅细胞聚集体的基于悬浮培养的策略，其促进了工艺开发和规模扩大。在hPSC培养基中形成聚集体4天，然后通过应用化学Wnt途径调节剂进行10天的定向分化。该方案适用于支持快速适应特定hPSC生产线要求的静态多孔格式。我们还演示了如何应用该协议使用搅拌罐生物反应器在100毫升的规模，提供了一个良好的控制放大平台CM生产。在生物反应器中，每个分化批次产生4000万至5000万个CM，纯度>80%，而无需进一步的谱系富集，可以在24天内实现。
Cardiomyocytes (CMs) generated from human pluripotent stem cells (hPSCs) are a potential cell source for regenerative therapies, drug discovery and disease modeling. All these applications require a routine supply of relatively large quantities of in vitro-generated CMs. This protocol describes a suspension culture-based strategy for the generation of hPSC-CMs as cell-only aggregates, which facilitates process development and scale-up. Aggregates are formed for 4 d in hPSC culture medium followed by 10 d of directed differentiation by applying chemical Wnt pathway modulators. The protocol is applicable to static multiwell formats supporting fast adaptation to specific hPSC line requirements. We also demonstrate how to apply the protocol using stirred tank bioreactors at a 100-ml scale, providing a well-controlled upscaling platform for CM production. In bioreactors, the generation of 40-50 million CMs per differentiation batch at >80% purity without further lineage enrichment can been achieved within 24 d.