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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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).
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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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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