Differentiation of human pluripotent stem cells to cardiomyocytes in scalable suspension culture

Developers

Henning Kempf, Christina Kropp, Ruth Olmer, Ulrich Martin, Robert Zweigerdt.

Description of the technology

Cardiomyocytes 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 cardiomyocytes.

This technology proposes protocol, which describes a suspension culture-based strategy for the generation of hPSC-cardiomyocytes 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 cardiomyocyte production. In bioreactors, the generation of 40–50 million cardiomyocytes per differentiation batch at >80% purity without further lineage enrichment can been achieved within 24 d.

The main advantage of suspension culture over 2D culture is that the aggregate-based culture approach circumvents the need for matrices that are typically required in 2D culture or for microcarrier-based suspension culture and differentiation, thereby reducing the number of culture components and supporting the achievement of conditions compliant with good manufacturing practices. A further advantage of the suspension approach is its compatibility with a wide range of culture platforms and respective process dimensions.

Practical application

The protocol according to this technology provides a universal prototype process that, at its current efficiency, could potentially be scaled up to 2,000 ml for the production of ~1×109 human cardiomyocytes in a single batch. The technology supports the systematic improvement of both cell expansion and cell differentiation efficiencies.

Although the first application of this technology is focused on hPSC-derived cardiomyocytes, the protocol could potentially be adapted to the mass production of other cell lineages, for example, hematopoietic cells, hepatocytes, pancreatic β-like cells and lung progenitor cells.

The technology is highly valuable for regenerative medicine, drug discovery, cell therapies, etc.

Laboratories

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

Links

Publications

• Kempf, H. et al. «Cardiac differentiation of human pluripotent stem cells in scalable suspension culture." 10 Nature Protocols (2015): 1345–1361.
• Kempf, H. et al. «Controlling expansion and cardiomyogenic differentiation of human pluripotent stem cells in scalable suspension culture." 3 Stem Cell Rep. (2014): 1132–1146.
• Zweigerdt, R., et al. «Scalable expansion of human pluripotent stem cells in suspension culture." 6 Nat. Protoc. (2011): 689–700.