Harnessing intermediate-scale bioreactors for next-generation macrophage production and application

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Harnessing intermediate-scale bioreactors for next-generation macrophage production and application

Data availability

We declare that the data supporting the findings of this study are available within the supporting Protocol13 and its Supplementary information. The scRNA sequencing datasets are deposited in the NCBI GEO repository under accession number GSE268458. Should any raw data files be needed in another format, they are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy (EXC 2155; RESIST; project no. 390874280 and DFG support LA 3680/9-1 and 10-1) (N.L.); the European Research Council (ERC) under the European Union (EU)’s Horizon 2020 research and innovation program (grant agreement 852178); and the EU (grant agreements 101100859 and 101158172) (N.L.). Additional funding was provided by the German Center of Lung Research (DZL) and the Federal Ministry of Research, Technology and Space (BMFTR, SMARTibone project). This work was supported by the Fraunhofer Internal Programs under grant no. attract 40-01696. The work also received funding by the SPARK BIH (01BIHTP2521B) funding scheme within the National Strategy for Gene- and Cell-based Therapies and by the program ‘zukunft.niedersachsen’ of Lower Saxony, Germany for the project ‘MacroAB-Delivery’. The views and opinions expressed are, however, those of the authors only and do not necessarily reflect those of the EU or the ERC. Neither the EU nor the granting authority can be held responsible for them. The project was additionally supported by zukunft.niedersachsen (Federal State of Lower Saxony), R2N.Micro-Replace-Systems. The EBiSC Bank acknowledges Bioneer A/S as the source of the human induced pluripotent cell line BIONi010-C, which was generated with support from the EBiSC project. The EBiSC has received support from the Innovative Medicines Initiative (IMI) Joint Undertaking (JU) under grant agreement no. 115582 and from the IMI-2 JU under grant agreement no. 821362, resources of which are composed of financial contributions from the European Union’s Seventh Framework Programme (FP7/2007–2013), the European Union’s Horizon 2020 research and innovation programme, and EFPIA.

Author information

Authors and Affiliations

  1. Department for Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School, Hannover, Germany

    Fawaz Saleh, Edwin Emilio Valdivia Malqui, Malene Kappelhøj, Eirini Nikolouli, Ariane Hai Ha Nguyen, Mi-Sun Jang, Débora Basílio-Queirós & Nico Lachmann

  2. Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, Hannover, Germany

    Ingrid Gensch, Maximilian Schinke & Nico Lachmann

  3. Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), German Center for Lung Research (DZL), Hannover, Germany

    Nico Lachmann

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

    Nico Lachmann

  5. Research Center for Translational Regenerative Medicine, Hannover Medical School, Hannover, Germany

    Nico Lachmann

Authors

  1. Fawaz Saleh
  2. Edwin Emilio Valdivia Malqui
  3. Ingrid Gensch
  4. Maximilian Schinke
  5. Malene Kappelhøj
  6. Eirini Nikolouli
  7. Ariane Hai Ha Nguyen
  8. Mi-Sun Jang
  9. Débora Basílio-Queirós
  10. Nico Lachmann

Contributions

Conceptualization: F.S. and N.L. Methodology: F.S. and N.L. Investigation: F.S., E.V.M., M.K., I.G., E.N., M.S., A.H.H.N., M.J., D.B.Q. and N.L. Writing—original draft: F.S., E.M.V., E.N., M.S., D.B.Q. and N.L. Writing—review and editing: F.S., E.M.V., M.K., I.G., E.N., M.S., A.H.H.N., M.J., D.B.Q. and N.L. Visualization: F.S., A.H.H.N. and N.L. Project administration: F.S. and N.L. Funding acquisition: N.L.

Corresponding author

Correspondence to Nico Lachmann.

Ethics declarations

Competing interests

N.L. is an author 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. N.L. is an author on the patent application (European patent application number PCT/EP2021/083371) entitled ‘Application of stem cell derived monocytes in a monocyte activation test for the assessment of pyrogenicity and inflammatory potential’. The priority date of the application is 29 November 2021. N.L. receives research funding from Novo Nordisk and holds a consultancy agreement with Evotec (scope outside the manuscript). All other authors declare no competing interests.

Peer review

Peer review information

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

Additional information

Key references

Ackermann, M. et al. Stem Cell Res. Ther. 15, 171 (2024): https://doi.org/10.1186/s13287-024-03785-2

Abdin, S. M. et al. J. Immunother. Cancer 11, e007705 (2023): https://doi.org/10.1136/jitc-2023-007705

Ackermann, M. et al. Nat. Protoc. 17, 513–539 (2022): https://doi.org/10.1038/s41596-021-00654-7

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

This Protocol is an extension to Nat. Protoc. 17, 513–539 (2022): https://doi.org/10.1038/s41596-021-00654-7

Extended data

Extended Data Fig. 1 Bioreactor process monitoring and assessment during mesoderm priming and macrophage production.

a) Representative graphs of bioreactor process parameters (CO2, pH, and temperature) during Mesoderm priming. b) Representative graphs of bioreactor process parameters (CO2, pH, and Temperature) during macrophage production. c) IL-6 secretion in naïve macrophages (iPSC lines: 1, 2, and 3). d) Phagocytosis of macrophages across different harvests 1b, 2, 3, 4, 5, 6, 7 (89.7 ± 7.3%, 84.8 ± 6.68%, 80.8 ± 6.03%, 81.2 ± 15.6%, 92.4 ± 7.97%, 95.2 ± 5.09%, SD +/- mean) (iPSC lines 1, 2, 3, 4, and 5, mean ± SD, n=10).

Extended Data Fig. 2 Morphological and cytological characterization of bioreactor generated iPSC-derived macrophages across multiple harvests and lines.

Representative of image of iPSC-derived macrophages produced in the benchtop bioreactor for iPSC 1, 2, 3, 4, and 5 at harvests 1a/1b, 3, 4 and 6. Top: brightfield, (magnification 10x, scale bare 100 µm) bottom: cytospin (magnification 20x) stained with May-Grünwald-Giemsa. Cytospin images were taken using Keyence BZ-X800 (Keyence, Japan) with 20x plan Achromat objective.

Extended Data Fig. 3 Single-cell transcriptomic profiling of iPSC-derived macrophages produced in bioreactors from various iPSC lines.

a) UMAP representation of dataset13 from three independent iMac harvests generated from CERO benchtop bioreactor, the cells are grouped by cell line. (iPSC lines 1, 2, and 3 were used). b) UMAP representation split by cell line and grouped by cluster identity. c) UMAP representation with normalized expression of hematopoietic/myeloid lineage marker genes (PTPRC, ITGAM, CD33, SPI1). d) Dot plot displaying the normalized expression of myeloid progenitor, macrophage, mast cell (MC), granulocyte (Gran), lymphoid lineage and fibroblast marker genes grouped by cell line (iPSC lines: 1-3). Panels a and b adapted from ref. 13, CC BY 4.0.

Extended Data Table 1 iPSC line details

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Supplementary information

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Saleh, F., Valdivia Malqui, E.E., Gensch, I. et al. Harnessing intermediate-scale bioreactors for next-generation macrophage production and application. Nat Protoc (2026). https://doi.org/10.1038/s41596-025-01313-x

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