ProtocolPublished: 22 June 2026Kevin Cyrys ORCID: orcid.org/0000-0001-5543-527X1,2,Felix Manstein ORCID: orcid.org/0000-0001-9740-96861,2,Wiebke Triebert ORCID: orcid.org/0000-0002-6927-14101,2,Nils Kriedemann ORCID: orcid.org/0000-0001-7767-15561,2,Carlos Alberto Hernandez-Bautista ORCID: orcid.org/0000-0003-2846-94441,2 &…Robert Zweigerdt ORCID: orcid.org/0000-0002-4656-07701,2 Nature Protocols (2026) Cite this articleSubjectsCell cultureRegenerative medicineStem-cell biotechnologyAbstractThe routine mass applications of human pluripotent stem (hPS) cell-derived progenies for regenerative medicine or high-throughput drug screenings will depend on the standardized supply of high-quality cells via controlled, efficient bioprocesses. Recent suspension culture (three-dimensional, 3D) strategies for hPS cell production in stirred-tank bioreactors (STBR) support this development. However, bioprocess inoculation still depends on adherent (two-dimensional, 2D) preculture, which is labor intensive, resource demanding and poorly controlled and limits process automation. Here we describe the controlled in-process production and dissociation of 3D cultured hPS cell aggregates directly in STBRs, tackling these challenges. The resulting cells can be used for the generation of high-density cryostocks, subsequently enabling the direct inoculation of 3D cultures, thereby entirely omitting the need for 2D preculture and the associated limitations. A key feature of this Protocol is the nonenzymatic, EDTA-based hPS cell aggregate dissociation approach in STBRs, enabling the impeller-based mechanical control of the dissociation process. The resulting cell suspension can be used for process reinoculation and seed train-based upscaling, as well as for the cryopreservation of produced hPS cells, ideally via controlled-rate freezing. Together, the protocol enables the efficient and flexible hPS cell suspension culture over multiple passages, maintaining karyotype stability and pluripotency. This Protocol can be easily implemented by any cell culture-educated scientist without extensive bioprocess training. Cell thawing requires 1 h, the 2D preculture requires 9 days, 2D cell passaging requires 1 h, bioreactor preparation requires 2 days, (direct) bioreactor inoculation requires 1.5 h, 3D STBR cultivation requires 3–4 days and STBR-based aggregate dissociation requires 2 h.Key pointsThis Protocol describes the controlled in-process production and dissociation of three-dimensional cultured human pluripotent stem cell aggregates, directly in stirred tank bioreactors. The resulting cells can be used for the process reinoculation, upscaling and generation of high-density cryostocks.The direct inoculation of three-dimensional cultures with high-density cryostocks overcomes the need for two-dimensional preculture, which is labor intensive, resource demanding and poorly controlled and limits closed-system processing and automation.This is a preview of subscription content, access via your institutionAccess optionsAccess Nature and 54 other Nature Portfolio journalsGet Nature+, our best-value online-access subscription27,99 € / 30 dayscancel any timeLearn moreSubscribe to this journalReceive 12 print issues and online access269,00 € per yearonly 22,42 € per issueLearn moreBuy this articlePurchase on SpringerLinkInstant access to the full article PDF.39,95 €Prices may be subject to local taxes which are calculated during checkoutFig. 1: Schematic outline of the described cultivation strategy.Fig. 2: Exemplary light microscopic pictures of hPS cell 2D monolayer cultures.Fig. 3: Bioreactor assembly.Fig. 4: Exemplary light microscopic pictures of suspension cultured hPS cells.Fig. 5: Anticipated results for the multipassage suspension cultivation inoculated froom 2D adherent culture-derived single hPS cells.Fig. 6: Anticipated results for the direct inoculation of STBR-based suspension culture with cryopreserved HDS of single hPS cells.Fig. 7: Anticipated results for the analysis of pluripotency, differentiation potential and karyotype stability.Data availabilityAll data included in this manuscript are available at https://doi.org/10.17605/OSF.IO/M7GKJ.Code availabilityThe scripts for the DASware are available in the Supplementary Code 1 (DASbox vessel script) and Supplementary Code 2 (Bioblock vessel script).ReferencesAckermann, M. et al. Continuous human Ipsc-macrophage mass production by suspension culture in stirred tank bioreactors. Nat. Protoc. 17, 513–539 (2022).CAS PubMed PubMed Central Google Scholar Kempf, H., Kropp, C., Olmer, R., Martin, U. & Zweigerdt, R. Cardiac differentiation of human pluripotent stem cells in scalable suspension culture. Nat. Protoc. 10, 1345–1361 (2015).CAS PubMed Google Scholar Kriedemann, N. et al. Standardized production of hPSC-derived cardiomyocyte aggregates in stirred spinner flasks. Nat. Protoc. 19, 1911–1939 (2024).Ilic, D. & Ogilvie, C. Pluripotent stem cells in clinical setting—new developments and overview of current status. Stem Cells 40, 791–801 (2022).PubMed PubMed Central Google Scholar Andrée, B. et al. Fabrication of heart tubes from iPSC derived cardiomyocytes and human fibrinogen by rotating mold technology. Sci. Rep. 14, 13174 (2024).PubMed PubMed Central Google Scholar Guo, L. et al. Estimating the risk of drug-induced proarrhythmia using human induced pluripotent stem cell–derived cardiomyocytes. Toxicol. Sci. 123, 281–289 (2011).CAS PubMed Google Scholar Medine, C. N. et al. Developing high-fidelity hepatotoxicity models from pluripotent stem cells. Stem Cells Transl. Med. 2, 505–509 (2013).CAS PubMed PubMed Central Google Scholar Kropp, C., Massai, D. & Zweigerdt, R. Progress and challenges in large-scale expansion of human pluripotent stem cells. Process Biochem. 59, 244–254 (2017).CAS Google Scholar Tannenbaum, S. E. & Reubinoff, B. E. Advances in hPSC expansion towards therapeutic entities: a review. Cell Prolif. 55, e13247 (2022).PubMed PubMed Central Google Scholar Abecasis, B. et al. Expansion of 3D human induced pluripotent stem cell aggregates in bioreactors: bioprocess intensification and scaling-up approaches. J. Biotechnol. 246, 81–93 (2017).CAS PubMed Google Scholar Huang, S. et al. Process development and scale-up of pluripotent stem cell manufacturing. Cell Gene Ther. Insights 6, 1277–1298 (2020).Google Scholar Kropp, C. et al. Impact of feeding strategies on the scalable expansion of human pluripotent stem cells in single-use stirred tank bioreactors. Stem Cells Transl. Med. 5, 1289–1301 (2016).PubMed PubMed Central Google Scholar Kwok, C. K. et al. Scalable stirred suspension culture for the generation of billions of human induced pluripotent stem cells using single-use bioreactors. J. Tissue Eng. Regen. Med. 12, 1076–1087 (2018).Google Scholar Manstein, F. et al. High density bioprocessing of human pluripotent stem cells by metabolic control and in silico modeling. Stem Cells Transl. Med. 10, 1063–1080 (2021).CAS PubMed PubMed Central Google Scholar Fan, Y., Wu, J., Ashok, P., Hsiung, M. & Tzanakakis, E. S. Production of human pluripotent stem cell therapeutics under defined xeno-free conditions: progress and challenges. Stem Cell Rev. Rep. 11, 96–109 (2015).CAS PubMed PubMed Central Google Scholar Wang, Y., Cheng, L. & Gerecht, S. Efficient and scalable expansion of human pluripotent stem cells under clinically compliant settings: a view in 2013. Ann. Biomed. Eng. 42, 1357–1372 (2014).CAS PubMed Google Scholar Ullmann, K. et al. Matrix-free human pluripotent stem cell manufacturing by seed train approach and intermediate cryopreservation. Stem Cell Res. Ther. 15, 89 (2024).CAS PubMed PubMed Central Google Scholar Konze, S. A. et al. Cleavage of E-cadherin and β-catenin by calpain affects Wnt signaling and spheroid formation in suspension cultures of human pluripotent stem cells. Mol. Cell. Proteom. 13, 990–1007 (2014).CAS Google Scholar Manstein, F., Ullmann, K., Triebert, W. & Zweigerdt, R. Process control and in silico modeling strategies for enabling high density culture of human pluripotent stem cells in stirred tank bioreactors. STAR Protoc. 2, 100988 (2021).CAS PubMed PubMed Central Google Scholar Pandey, P. R. et al. End-to-end platform for human pluripotent stem cell manufacturing. Int. J. Mol. Sci. 21, 89 (2020).CAS Google Scholar Borys, B. S. et al. Overcoming bioprocess bottlenecks in the large-scale expansion of high-quality hiPSC aggregates in vertical-wheel stirred suspension bioreactors. Stem Cell Res. Ther. 12, 55 (2021).CAS PubMed PubMed Central Google Scholar Cuesta-Gomez, N. et al. Suspension culture improves Ipsc expansion and pluripotency phenotype. Stem Cell Res. Ther. 14, 154 (2023).CAS PubMed PubMed Central Google Scholar Davis, B. M., Loghin, E. R., Conway, K. R. & Zhang, X. Automated closed-system expansion of pluripotent stem cell aggregates in a rocking-motion bioreactor. SLAS Technol. 23, 364–373 (2018).CAS PubMed Google Scholar Borys, B. S. et al. Robust bioprocess design and evaluation of commercial media for the serial expansion of human induced pluripotent stem cell aggregate cultures in vertical-wheel bioreactors. Stem Cell Res. Ther. 15, 232 (2024).CAS PubMed PubMed Central Google Scholar Beers, J. et al. Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions. Nat. Protoc. 7, 2029–2040 (2012).CAS PubMed PubMed Central Google Scholar Rodin, S. et al. Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment. Nat. Commun. 5, 3195 (2014).PubMed Google Scholar Tohyama, S. et al. Efficient large-scale 2D culture system for human induced pluripotent stem cells and differentiated cardiomyocytes. Stem Cell Rep. 9, 1406–1414 (2017).CAS Google Scholar Lam, A. T.-L., Chen, A. K.-L., Ting, S. Q.-P., Reuveny, S. & Oh, S. K.-W. Integrated processes for expansion and differentiation of human pluripotent stem cells in suspended microcarriers cultures. Biochem. Biophys. Res. Commun. 473, 764–768 (2016).CAS PubMed Google Scholar Ting, S., Chen, A., Reuveny, S. & Oh, S. An intermittent rocking platform for integrated expansion and differentiation of human pluripotent stem cells to cardiomyocytes in suspended microcarrier cultures. Stem Cell Res. 13, 202–213 (2014).CAS PubMed Google Scholar Fonoudi, H., Lyra-Leite, D. M., Javed, H. A. & Burridge, P. W. Generating a cost-effective, weekend-free chemically defined human induced pluripotent stem cell (hiPSC) culture medium. Curr. Protoc. Stem Cell Biol. 53, e110 (2020).CAS PubMed PubMed Central Google Scholar Lyra-Leite, D. M. et al. Nutritional requirements of human induced pluripotent stem cells. Stem Cell Rep. 18, 1371–1387 (2023).CAS Google Scholar Clarke, D., Norris, D. & Bennett, B. Development of a novel thermoplastic tubing, FP-FLEX™, and single-use freezing bag for working cell banks enabling closed-system processing to temperatures as low as −196°C. BMC Proc. 9, P58 (2015).PubMed Central Google Scholar Chen, Y. et al. A versatile polypharmacology platform promotes cytoprotection and viability of human pluripotent and differentiated cells. Nat. Methods 18, 528–541 (2021).CAS PubMed PubMed Central Google Scholar Xu, Y. et al. Revealing a core signaling regulatory mechanism for pluripotent stem cell survival and self-renewal by small molecules. Proc. Natl Acad. Sci. USA 107, 8129–8134 (2010).CAS PubMed PubMed Central Google Scholar Sen, A., Kallos, M. S. & Behie, L. A. Effects of hydrodynamics on cultures of mammalian neural stem cell aggregates in suspension bioreactors. Ind. Eng. Chem. Res. 40, 5350–5357 (2001).CAS Google Scholar Wu, J., Rostami, M. R., Cadavid Olaya, D. P. & Tzanakakis, E. S. Oxygen transport and stem cell aggregation in stirred-suspension bioreactor cultures. PloS ONE 9, e102486 (2014).PubMed PubMed Central Google Scholar Hartung, S. et al. Directing cardiomyogenic differentiation of human pluripotent stem cells by plasmid-based transient overexpression of cardiac transcription factors. Stem Cells Dev. 22, 1112–1125 (2013).CAS PubMed PubMed Central Google Scholar Haase, A. et al. GMP-compatible manufacturing of three Ips cell lines from human peripheral blood. Stem Cell Res. 35, 101394 (2019).CAS PubMed Google Scholar Davis, R. P. et al. Targeting a GFP reporter gene to the MIXL1 locus of human embryonic stem cells identifies human primitive streak–like cells and enables isolation of primitive hematopoietic precursors. Blood 111, 1876–1884 (2008).CAS PubMed Google Scholar Chen, G. et al. Chemically defined conditions for human Ipsc derivation and culture. Nat. Methods 8, 424–429 (2011).CAS PubMed PubMed Central Google Scholar Zweigerdt, R., Olmer, R., Singh, H., Haverich, A. & Martin, U. Scalable expansion of human pluripotent stem cells in suspension culture. Nat. Protoc. 6, 689–700 (2011).CAS PubMed Google Scholar Ben-David, U. et al. Aneuploidy induces profound changes in gene expression, proliferation and tumorigenicity of human pluripotent stem cells. Nat. Commun. 5, 4825 (2014).CAS PubMed Google Scholar Lezmi, E., Jung, J. & Benvenisty, N. High prevalence of acquired cancer-related mutations in 146 human pluripotent stem cell lines and their differentiated derivatives. Nat. Biotechnol. 42, 1667–1671 (2024).CAS PubMed Google Scholar Download referencesAcknowledgementsWe thank G. Göhring (Amedes Genetics) and the Department of Human Genetics at Hannover Medical School for their support with the karyotype analysis.FundingThis work was supported by grants to R.Z. including: German Research Foundation (DFG; grant nos. Cluster of Excellence REBIRTH EXC 62/2 and ZW64/4-2), Federal Ministry of Education and Research (BMBF; grant nos. 01EK1601A, 031L0249 and 01EK2108A), Lower Saxony (‘Zukunft Niedersachsen’ grant no. ZN4092, ‘Förderung aus EFRE-Mitteln’ grant no. ZW3-87035144), NCR3s UK (grant no. NC/Z500707/1) and the European Union (EU Horizon, project HEAL, contract no. 101056712); the authors explicitly express that funding under contract no. 101056712 was used for research on human induced pluripotent stem cells only in frame of the here-published research. 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.Author informationAuthors and AffiliationsLeibniz Research Laboratories for Biotechnology and Artificial Organs, Department of Cardiothoracic Transplantation and Vascular Surgery, Hannover Medical School, Hannover, GermanyKevin Cyrys, Felix Manstein, Wiebke Triebert, Nils Kriedemann, Carlos Alberto Hernandez-Bautista & Robert ZweigerdtREBIRTH Research Center for Translational and Regenerative Medicine, Hannover Medical School, Hannover, GermanyKevin Cyrys, Felix Manstein, Wiebke Triebert, Nils Kriedemann, Carlos Alberto Hernandez-Bautista & Robert ZweigerdtAuthorsKevin CyrysView author publicationsSearch author on:PubMed Google ScholarFelix MansteinView author publicationsSearch author on:PubMed Google ScholarWiebke TriebertView author publicationsSearch author on:PubMed Google ScholarNils KriedemannView author publicationsSearch author on:PubMed Google ScholarCarlos Alberto Hernandez-BautistaView author publicationsSearch author on:PubMed Google ScholarRobert ZweigerdtView author publicationsSearch author on:PubMed Google ScholarContributionsK.C., F.M. and R.Z.: developed the protocol. K.C.: collection and analysis of data and data interpretation. F.M., W.T., N.K. and C. H.-B.: collection of data. F.M.: DASware script writing. K.C. and R.Z.: article writing.Corresponding authorsCorrespondence to Kevin Cyrys or Robert Zweigerdt.Ethics declarationsCompeting interestsThe authors declare no competing interests.Peer reviewPeer review informationNature Protocols thanks Michael Kallos (who co-reviewed with Tiffany Dang), Yan Li and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Key referencesManstein, F. et al. Stem Cells Transl. Med. 10, 1063–1080 (2021): https://doi.org/10.1002/sctm.20-0453Ullmann, K. et al. Stem Cell Res. Ther. 15, 89 (2024): https://doi.org/10.1186/s13287-024-03699-zExtended dataExtended Data Fig. 1 Process data for direct inoculation approaches with the HES3 MIXL1–GFP and the GMPDU_8 cell lines.(a–d) Process data for a direct inoculation approach with the HES3 MIXL1–GFP cell line at 1 L process scale cultivated for 4 d (n = 1). (a) The viable cell density (bar graph) and the cell viability (line graph). (b) The specific growth rate µ. (c) The aggregate diameter distribution. (d) Exemplary light microscopic pictures of process-derived HES3 MIXL1–GFP aggregates. (e–h) Process data for a direct inoculation approach with the GMPDU_8 cell line at 150 mL process scale for 3 consecutive passages (n = 3; MEAN ± SEM). (e) The viable cell density (bar graph) and the cell viability (line graph). (f) The specific growth rate µ. (g) The aggregate diameter distribution. (h) Exemplary light microscopic pictures of process-derived GMPDU_8 aggregates over 3 passages.Supplementary informationSupplementary Information (download PDF )Supplementary Figs. 1–6, Supplementary Tables 1 and 2 and Supplementary Methods 1 and 2.Supplementary Code 1 (download PDF )DASbox vessel script.Supplementary Code 2 (download PDF )Bioblock vessel script.Rights and permissionsSpringer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.Reprints and permissionsAbout this article