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Continuous human iPSC-macrophage mass production by suspension culture in stirred tank bioreactors

Nature Protocols volume 17, pages 513–539 (2022)Cite this article

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Abstract

Macrophages derived from human induced pluripotent stem cells (iPSCs) have the potential to enable the development of cell-based therapies for numerous disease conditions. We here provide a detailed protocol for the mass production of iPSC-derived macrophages (iPSC-Mac) in scalable suspension culture on an orbital shaker or in stirred-tank bioreactors (STBRs). This strategy is straightforward, robust and characterized by the differentiation of primed iPSC aggregates into ‘myeloid-cell-forming-complex’ intermediates by means of a minimal cytokine cocktail. In contrast to the ‘batch-like differentiation approaches’ established for other iPSC-derived lineages, myeloid-cell-forming-complex-intermediates are stably maintained in suspension culture and continuously generate functional and highly pure iPSC-Mac. Employing a culture volume of 120 ml in the STBR platform, ~1–4 × 107 iPSC-Mac can be harvested at weekly intervals for several months. The STBR technology allows for real-time monitoring of crucial process parameters such as biomass, pH, dissolved oxygen, and nutrition levels; the system also promotes systematic process development, optimization and linear upscaling. The process duration, from the expansion of iPSC until the first iPSC-Mac harvest, is 28 d. Successful application of the protocol requires expertise in pluripotent stem cell culture, differentiation and analytical methods, such as flow cytometry. Fundamental know-how in biotechnology is also advantageous to run the process in the STBR platform. The continuous, scalable production of well-defined iPSC-Mac populations is highly relevant to various fields, ranging from developmental biology, immunology and cell therapies to industrial applications for drug safety and discovery.

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Fig. 1: Schematic representation of the differentiation platform.
Fig. 2: Morphology of human iPSCs in 2D culture.
Fig. 3: Morphology of primed aggregates.
Fig. 4: Photography of the assembled bioreactor (related to Box 1).
Fig. 5: Production of iPSC-Mac by MCFCs.
Fig. 6: Harvest efficiency and process monitoring of the STBR production platform.
Fig. 7: Analysis of iPSC-Mac by flow cytometry (related to Box 4).
Fig. 8: Phagocytosis of bioparticles by iPSC-Mac (related to Box 5).

Data availability statement

The raw data for Figs. 7 and 8 are available under FlowRepository IDs FR-FCM-Z4WC (Fig. 7) and FR-FCM-Z4WE (Fig. 8)). Source data are provided with this paper.

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Acknowledgements

The authors thank D. Kloos, T. Buchegger and L. Bach for technical support. The work received funding from the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG): Cluster of Excellence REBIRTH EXC 62/3 (R.Z. and N.L.), ZW64/4-1, ZW 64/4-2, KFO311/ZW64/7-1 (R.Z.); the German Ministry for Education and Science (Bundesministerium für Bildung und Forschung, BMBF): 01EK1602A (R.Z. and N.L.) 13N14086, 01EK1601A, 655 13XP5092B, 031L0249 (R.Z.), ‘Förderung aus Mitteln des Niedersächsischen Vorab’ (grant ZN3340) (R.Z. and N.L.), the Else Kröner-Fresenius-Stiftung (EKFS; 2016_A146) (M.A.). The work also received funding from Hannover Medical School Transplantation Center (Tx Center) (N.L.) and the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) (N.L.). This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 852178).

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Author notes

  1. These authors contributed equally: Mania Ackermann, Anna Rafiei Hashtchin, Robert Zweigerdt, Nico Lachmann.

Authors and Affiliations

  1. Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany

    Mania Ackermann, Anna Rafiei Hashtchin, Marco Carvalho Oliveira & Nico Lachmann

  2. REBIRTH, Research Center for Translational and Regenerative Medicine, Hannover Medical School, Hannover, Germany

    Mania Ackermann, Anna Rafiei Hashtchin, Felix Manstein, Marco Carvalho Oliveira, Robert Zweigerdt & Nico Lachmann

  3. Department of Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School, Hannover, Germany

    Mania Ackermann & Nico Lachmann

  4. Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, Germany

    Felix Manstein, Henning Kempf & Robert Zweigerdt

  5. Department of Stem Cell Biology, Novo Nordisk A/S, Måløv, Denmark

    Henning Kempf

  6. Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Center for Lung Research (DZL), Hannover, Germany

    Nico Lachmann

  7. Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, Hannover, Germany

    Nico Lachmann

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Contributions

M.A., H.K., R.Z. and N.L. developed the protocol; M.A., A.R.H. and F.M. performed the experiments and analyzed the data; M.A., A.R.H., F.M., M.C.O., R.Z. and N.L wrote the manuscript; all authors approved the final paper.

Corresponding author

Correspondence to Nico Lachmann.

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Competing interests

This work is included in a patent application. M.A., H.K., R.Z. and N.L. are authors of the patent application (European patent application number PCT/EP2018/061574) entitled ‘Stem-cell derived myeloid cells, generation and use thereof’. The priority date of the application is 4 May 2017. H.K. is an employee of Novo Nordisk A/S, Måløv, Denmark, and N.L. and R.Z. receive funding from Novo Nordisk A/S, Måløv, Denmark.

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Nature Protocols thanks Dan Kaufman, Lesley Forrester and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Ackermann, M. et al. Nat. Commun. 9, 5088 (2018): https://doi.org/10.1038/s41467-018-07570-7

Ackermann, M et al. Haematologica 106, 1354–1367 (2021): https://doi.org/10.3324/haematol.2019.228064

Rafiei Hashtchin, A. et al. Blood Adv. 5, 5190–5201 (2021): https://doi.org/10.1182/bloodadvances.2021004853

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Ackermann, M., Rafiei Hashtchin, A., Manstein, F. et al. Continuous human iPSC-macrophage mass production by suspension culture in stirred tank bioreactors. Nat Protoc 17, 513–539 (2022). https://doi.org/10.1038/s41596-021-00654-7

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