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Nat Protoc. 2015 Sep;10(9):1345-61. doi: 10.1038/nprot.2015.089. Epub 2015 Aug 13.

Cardiac differentiation of human pluripotent stem cells in scalable suspension culture.

Author information

1
1] Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiac, Thoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, Germany. [2] REBIRTH-Cluster of Excellence, Hannover Medical School, Hannover, Germany.
2
1] Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiac, Thoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, Germany. [2] REBIRTH-Cluster of Excellence, Hannover Medical School, Hannover, Germany. [3] Member of Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Center for Lung Research (DZL), Hannover, Germany.

Abstract

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.

PMID:
26270394
DOI:
10.1038/nprot.2015.089
[Indexed for MEDLINE]

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