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Standardized production of hPSC-derived cardiomyocyte aggregates in stirred spinner flasks

Nature Protocols (2024)Cite this article

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Abstract

A promising cell-therapy approach for heart failure aims at differentiating human pluripotent stem cells (hPSCs) into functional cardiomyocytes (CMs) in vitro to replace the disease-induced loss of patients’ heart muscle cells in vivo. But many challenges remain for the routine clinical application of hPSC-derived CMs (hPSC-CMs), including good manufacturing practice (GMP)-compliant production strategies. This protocol describes the efficient generation of hPSC-CM aggregates in suspension culture, emphasizing process simplicity, robustness and GMP compliance. The strategy promotes clinical translation and other applications that require large numbers of CMs. Using a simple spinner-flask platform, this protocol is applicable to a broad range of users with general experience in handling hPSCs without extensive know-how in biotechnology. hPSCs are expanded in monolayer to generate the required cell numbers for process inoculation in suspension culture, followed by stirring-controlled formation of cell-only aggregates at a 300-ml scale. After 48 h at checkpoint (CP) 0, chemically defined cardiac differentiation is induced by WNT-pathway modulation through use of the glycogen-synthase kinase-3 inhibitor CHIR99021 (WNT agonist), which is replaced 24 h later by the chemical WNT-pathway inhibitor IWP-2. The exact application of the described process parameters is important to ensure process efficiency and robustness. After 10 d of differentiation (CP I), the production of ≥100 × 106 CMs is expected. Moreover, to ‘uncouple’ cell production from downstream applications, continuous maintenance of CM aggregates for up to 35 d in culture (CP II) is demonstrated without a reduction in CM content, supporting downstream logistics while potentially overcoming the requirement for cryopreservation.

Key points

  • We present a protocol for the efficient generation of hPSC-CM aggregates in suspension culture, emphasizing process simplicity, robustness and GMP compliance. The strategy promotes clinical translation and other applications that require large numbers of CMs.

  • This protocol uses a simple spinner-flask platform, making it accessible to users experienced in the handling of hPSCs but without extensive experience in biotechnology. This enables straightforward adaptation by many laboratories without bioprocessing experience.

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Fig. 1: Schematic display of key steps and timelines of the protocol and the applied culture strategy and vessels.
Fig. 2: Typical appearance of a sub-confluent hPSC culture (hiPSC line Phoenix) grown for 3 d in monolayer before splitting on respective culture matrices.
Fig. 3: Exemplified results expected for quality assurance at CP 0.
Fig. 4: Establishing a viability assay for whole aggregate analysis.
Fig. 5: Typical outcome of QC assessment at CP I and CP II.
Fig. 6: Flow cytometry–based analysis of cells not expressing cTnT (‘non-cardiomyocytes’) at CP I.

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Data availability

The flow cytometry data are available in the FlowRepository under accession code FR-FCM-Z6K3. All remaining data generated or analyzed during this study are included in this published article and its supplementary files.

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Acknowledgements

This work was supported by the German Research Foundation (DFG; grants Cluster of Excellence REBIRTH EXC 62/2 and ZW64/4-2), the Federal Ministry of Education and Research (BMBF; grants 01EK1601A, 13XP5092B, 031L0249 and 01EK2108A), Lower Saxony ‘Förderung aus Mitteln des Niedersächsischen Vorab’ (grant ZN3340) and ‘Niedersächsische Ministerium für Wissenschaft und Kultur’ (MWK; grant ZN4092) and the European Union (Horizon Europe project HEAL grant 101056712). The views and opinions expressed are, however, those of the authors only and do not necessarily reflect those of the European Union or the European Health and Digital Executive Agency (HADEA). Neither the European Union nor the granting authority can be held responsible for them. We thank R. Bauerfeind and O. Terwolbeck from the MHH Core Unit for laser microscopy and for help with confocal microscopy.

Author information

Author notes

  1. These authors contributed equally: Nils Kriedemann, Wiebke Triebert.

Authors and Affiliations

  1. Department of Cardiothoracic, Transplantation and Vascular Surgery (HTTG), Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO); REBIRTH–Research Center for Translational Regenerative Medicine; Hannover Medical School (MHH), Hannover, Germany

    Nils Kriedemann, Wiebke Triebert, Jana Teske, Mira Mertens, Annika Franke, Kevin Ullmann, Felix Manstein, Lika Drakhlis, Alexandra Haase, Caroline Halloin, Ulrich Martin & Robert Zweigerdt

  2. Evotec, Hamburg, Germany

    Wiebke Triebert & Felix Manstein

  3. Department of Cell Therapy Process Technology, Novo Nordisk, Måløv, Denmark

    Caroline Halloin

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Contributions

N.K., W.T., C.H., U.M. and R.Z. designed the experiments. N.K., W.T., C.H., M.M., A.F., L.D. and J.T. contributed to the experimental design, performed the experiments and analyzed the data. A.H. generated hiPSC lines. F.M., K.U. and C.H. developed scripts for automatized analysis of the experiments. N.K., W.T., U.M. and R.Z. wrote and reviewed the manuscript.

Corresponding authors

Correspondence to Nils Kriedemann or Robert Zweigerdt.

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Competing interests

C.H. is an employee of Novo Nordisk. F.M. and W.T. are employees of Evotec. The other authors declare no competing interests.

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Nature Protocols thanks Kurt Pfannkuche and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Key reference using this protocol

Halloin, C. et al. Stem Cell Rep. 13, 366–379 (2019): https://doi.org/10.1016/j.stemcr.2019.09.001

Extended data

Extended Data Fig. 1 Exemplary production of hiPSC-derived cardiomyocytes from vitronectin and (GMP-conforming) CTS vitronectin pre-culture.

a and b, Appearance of cells on CTS vitronectin (a) and vitronectin (b) on day −2 of the production protocol in monolayer (scale bar, 500 µm). c, Expression of markers of an undifferentiated state at CP 0 before induction of differentiation in samples from spinner flasks pre-cultured on vitronectin or CTS vitronectin. d, Cell yield at CP I of respective spinner-flask runs. e, Aggregate diameter of representative samples taken at CP I from respective spinners. Shown are individual values and mean ± s.d. in red. f, Percentage of cells positive for cardiac markers in representative samples from the respective spinner flasks at CP I.

Extended Data Fig. 2 Setup of the spinner flask.

In a sterile environment, unpack from plastic and install the lid with the stirrer. Remove one of the lids for the side ports and install a sampling device (optional).

Extended Data Fig. 3 Aggregate diameter size distribution for one differentiation run at CP 0, CP I and CP II.

Shown are individual aggregate diameters (gray) and mean values ± s.d. (red). The hiPSC line used is Amber.

Extended Data Fig. 4 Total aggregate count per milliliter of spinner flask experiments at CP 0 (n = 3) and CP I (n = 2).

Aggregate numbers were determined through dilution of a culture sample and manual counting through microscopy.

Extended Data Fig. 5 Confocal microscopy of aggregates at CP 0 and CP I to analyze cell number per aggregate.

a–c, Light microscope pictures of a representative cell sample at CP 0 (scale bar, 500 µm) (a) and confocal images of the central Z-stack of aggregates of the same batch (scale bar, 50 µm) (b and c). d–f, Light microscope pictures of a representative cell sample at CP I (scale bar, 500 µm) (d) and confocal images in a differentiated state as CM aggregates at CP I (scale bar, 50 µm) (e and f). Nuclei were stained with Sytox red after fixation; afterwards, aggregates were dehydrated with ethanol and cleared with methyl salicylate/benzyl benzoate (MSBB). Notable are pronounced cavities at CP 0 as well as at CP I. g, Aggregates treated for confocal imaging do not differ in diameter compared to untreated, equivalent samples measured according to QC3 at CP 0 or CP I. h, Nuclei identified at CP 0 and CP I through automatic counting of confocal z-stacks imaging whole aggregates. This nuclei count should closely resemble the total cell count per aggregate. i and j, Identified nuclei per aggregate in comparison to the individual aggregate diameter for aggregates at CP 0 (i) and CP I (j). Linear regression was added to depict the goodness of fit (CP 0 R2= 0.73; CP I R2= 0.55). For examples, see supplementary file z-stacks for CP 0 and CP I (open with the hyperstack function of FIJI/ImageJ). PFA, paraformaldehyde. The hiPSC line Phoenix was used in all experiments.

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2

Supplementary Video 1

CM harvest for downstream application

Supplementary Video 2

hPSC splitting

Supplementary Video 3

Spinner medium exchange

Supplementary Data 1

Confocal z-stack pluripotent aggregates CP 0

Supplementary Data 2

Confocal z-stack CM aggregates CP I

Supplementary Code 1

ImageJ macro for aggregate size determination

Supplementary Code 2

ImageJ macro for viability score determination

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Kriedemann, N., Triebert, W., Teske, J. et al. Standardized production of hPSC-derived cardiomyocyte aggregates in stirred spinner flasks. Nat Protoc (2024). https://doi.org/10.1038/s41596-024-00976-2

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