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

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. Access through your institution Buy or subscribe This is a preview of

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* Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS EFFICIENT AND REPRODUCIBLE GENERATION OF HUMAN

IPSC-DERIVED CARDIOMYOCYTES AND CARDIAC ORGANOIDS IN STIRRED SUSPENSION SYSTEMS Article Open access 15 July 2024 A MINIATURE DIALYSIS-CULTURE DEVICE ALLOWS HIGH-DENSITY HUMAN-INDUCED

PLURIPOTENT STEM CELLS EXPANSION FROM GROWTH FACTOR ACCUMULATION Article Open access 19 November 2021 PRODUCTION AND CRYOPRESERVATION OF DEFINITIVE ENDODERM FROM HUMAN PLURIPOTENT STEM CELLS

UNDER DEFINED AND SCALABLE CULTURE CONDITIONS Article 12 February 2021 DATA AVAILABILITY The flow cytometry data are available in the FlowRepository under accession code FR-FCM-Z6K3. All

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from human iPS cells. _Stem Cell Res. Ther._ 13, 251 (2022). Article  CAS  PubMed  PubMed Central  Google Scholar  Download references 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 * These authors contributed equally: Nils Kriedemann, Wiebke

Triebert. AUTHORS AND AFFILIATIONS * 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 * Evotec, Hamburg, Germany Wiebke Triebert & Felix Manstein *

Department of Cell Therapy Process Technology, Novo Nordisk, Måløv, Denmark Caroline Halloin Authors * Nils Kriedemann View author publications You can also search for this author inPubMed 

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author inPubMed Google Scholar * Mira Mertens View author publications You can also search for this author inPubMed Google Scholar * Annika Franke View author publications You can also

search for this author inPubMed Google Scholar * Kevin Ullmann View author publications You can also search for this author inPubMed Google Scholar * Felix Manstein View author publications

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publications You can also search for this author inPubMed Google Scholar * Caroline Halloin View author publications You can also search for this author inPubMed Google Scholar * Ulrich

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Scholar 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. ETHICS DECLARATIONS 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. PEER REVIEW PEER REVIEW INFORMATION _Nature Protocols_ thanks Kurt Pfannkuche and the other, anonymous reviewer(s) for

their contribution to the peer review of this work. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and

institutional affiliations. RELATED LINKS 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 _R_2= 0.73; CP I _R_2= 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

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