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Human induced pluripotent stem cell-derived cardiomyocytes and their use in a cardiac organ-on-a-chip to assay electrophysiology, calcium and contractility

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Abstract

Cardiac organs-on-a-chip (OoCs) or microphysiological systems have the potential to predict cardiac effects of new drug candidates, including unanticipated cardiac outcomes, which are among the main causes for drug attrition. This protocol describes how to prepare and use a cardiac OoC containing cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS cells). The use of cells derived from hiPS cells as reliable sources of human cells from diverse genetic backgrounds also holds great potential, especially when cultured in OoCs that are physiologically relevant culture platforms. To promote the broad adoption of hiPS cell-derived cardiac OoCs in the drug development field, there is a need to first ensure reproducibility in their preparation and use. This protocol aims to provide key information on how to reduce sources of variability during hiPS cell maintenance, differentiation, loading and maturation in OoCs. Variability in these procedures can lead to inconsistent purity after differentiation and variable function between batches of microtissues formed in OoCs. This protocol also focuses on describing the handling and functional assessment of cardiac microtissues using live-cell microscopy approaches to quantify parameters of cellular electrophysiology, calcium transients and contractility. The protocol consists of five stages: (1) thaw and maintain hiPS cells, (2) differentiate hiPS cell cardiomyocytes, (3) load differentiated cells into OoCs, (4) maintain and characterize loaded cells, and (5) evaluate and utilize cardiac OoCs. Execution of the entire protocol takes ~40 days. The required skills to carry out the protocol are experience with sterile techniques, mammalian cell culture and maintaining hiPS cells in a pluripotent state.

Key points

  • Preparation and use of a cardiac organ-on-a-chip is described in five stages: thawing of human induced pluripotent stem cells, differentiation into cardiomyocytes, loading the microfluidic chip with cardiomyocytes, maturation of microtissues and functional characterization with image-based assays.

  • This protocol reproduces the physiology of the myocardium more robustly than traditional 2D cultures, ensuring reproducibility and reducing variability in results that can limit the use of organs-on-a-chip in drug development.

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Fig. 1: Layout of plates used after thawing, while maintaining and before differentiating WTC11 GCaMP6f-hiPS cells and morphological features of cells in culture.
Fig. 2: Variations in the morphology of cells in culture during the three days that preceded the onset of differentiation and the first six days of differentiation of hiPS cells into cardiomyocytes.
Fig. 3: Loading differentiated cells into cardiac OoCs.
Fig. 4: Two centrifugation steps used to load differentiated hiPS cell-cardiomyocytes into the cell chamber of the OoC.
Fig. 5: Timeline of the preparation stages of OoCs after differentiating cardiomyocytes.
Fig. 6: Variations in the cardiac function of OoCs were recorded in response to changes in extracellular calcium concentration (0.125–2 mM).
Fig. 7: Beat rate-dependent functional output of OoCs within the range of 0.75–2 Hz.

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Data availability

Data supporting the findings of this study are shared in the paper. Source data accompany this paper. Source data are provided with this paper.

Code availability

Codes used for analyzing videos of fluorescent labels in cells are publicly available at Zenodo https://doi.org/10.5281/zenodo.12563741. The graphical user interface for characterizing movement of cells with digital image correlation using brightfield microscopy videos of beating cells has been published and is publicly available18.

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Acknowledgements

The authors thank J. Florian from the Division of Applied Regulatory Science at the US Food and Drug Administration (FDA) for his valuable suggestions during the writing of the manuscript; E. Miller (Berkeley College of Chemistry) for supplying the voltage sensing dye BeRST; J. Berger from the FDA Library for her editorial support, and Z. Ma for his valuable assistance during the manuscript preparation.

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Authors and Affiliations

  1. Division of Applied Regulatory Science, Office of Clinical Pharmacology, Office of Translational Sciences, Center for Drug Evaluation and Research, US Food and Drug Administration, Silver Spring, MD, USA

    M. Iveth Garcia, Keri Dame, Bhavya Bhardwaj, Ryosuke Yokosawa, Dylan Bruckner, Kevin A. Ford & Alexandre J. S. Ribeiro

  2. Department of Bioengineering and California Institute for Quantitative Biosciences (QB3), University of California at Berkeley, Berkeley, CA, USA

    Verena Charwat, Brian A. Siemons, Ishan Goswami & Kevin E. Healy

  3. Department of Computational Physiology, Simula Research Laboratory, Oslo, Norway

    Henrik Finsberg & Samuel T. Wall

  4. Booz Allen Hamilton, McLean, VA, USA

    Dylan Bruckner

  5. Department of Materials Science and Engineering, University of California at Berkeley, Berkeley, CA, USA

    Kevin E. Healy

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Contributions

M.I.G., A.J.S.R., K.D., V.C., R.Y. and B.A.S. developed and optimized the protocol. M.I.G. and A.J.S.R. wrote the manuscript. M.I.G. performed experiments. V.C., B.A.S. and K.E.H. provided microfluidic chambers and protocols for handling and use. H.F. and S.T.W. contributed to the development of software used for this protocol. All authors contributed to the manuscript and approved the submitted version.

Corresponding authors

Correspondence to M. Iveth Garcia or Alexandre J. S. Ribeiro.

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Competing interests

Work for the manuscript was completed by A.J.S.R., K.D., and R.Y. while employees of the FDA. A.J.S.R is currently employed by Hovione PharmaScience Ltd. K.D. is currently employed by United Therapeutics Corporation. R.Y. is currently part of the Department of Biomedical Engineering and Mechanics at Virginia Polytechnic Institute and State University. K.E.H., V.C., S.T.W., H.F. and B.A.S. have either a financial or equity relationship with Organos Inc. and both they and the company may benefit from commercialization of the results of this research. All other authors declare that they have no competing financial interests and that the article reflects the views of the authors and should not be construed to represent the FDA’s views or policies.

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Nature Protocols thanks Brian Timko, Hidetoshi Masumoto, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references

Mathur, A. et al. Sci. Rep. 5, 8883 (2015): https://doi.org/10.1038/srep08883

Charrez, B. et al. Front. Pharmacol. 12, 684252 (2021): https://doi.org/10.3389/fphar.2021.684252

Huebsch, N. et al. Nat. Biomed. Eng. 6, 372–388 (2022): https://doi.org/10.1038/s41551-022-00884-4

Arefin, A. et al. Front. Pharmacol. 14, 1212092 (2023): https://doi.org/10.3389/fphar.2023.1212092

Supplementary information

Supplementary Information

Section 1. Supplementary Figs. 1–4, Section 2. Supplementary Tables 1 and 2, Section 3. Supplementary Methods, Section 4. Supplementary Software. Section 5. Supplementary References.

Supplementary Video 1

Brightfield video of differentiated hiPS cell-cardiomyocytes. Cardiomyocyte contractions were first observed by light microscopy at day 7 of differentiation and were easily observed by naked eye without microscopic magnification from day 8 onward. Sheet-like appearance is expected to start spontaneously contract as a single sheet starting day 7 of differentiation.

Supplementary Video 2

Video of differentiated hiPS cell-cardiomyocytes expressing GCaMP6f fluorescent protein. For cells expressing GCaMP6f, changes in fluorescent intensity are typically apparent. Using eGFP channel, intracellular calcium typically seems to flow evenly within the beating sheet of cells. Fluorescent variations usually start being observed on day 7 of differentiation.

Supplementary Video 3

Video example of over excited tissue before and after 20 min rest. Representative videos showing over excited tissue after handling.

Supplementary Video 4

Video example of over excited tissue before and after 20 min rest. Representative video shows how tissue goes back to baseline spontaneous contraction after 20 min rest on the microscope.

Source data

Source Data Fig. 6

Source data for contraction, intracellular calcium, action potential and raw data for calcium trace.

Source Data Fig. 7

Source data for contraction, intracellular calcium, action potential and raw data for contraction displacement trace.

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Garcia, M.I., Dame, K., Charwat, V. et al. Human induced pluripotent stem cell-derived cardiomyocytes and their use in a cardiac organ-on-a-chip to assay electrophysiology, calcium and contractility. Nat Protoc (2025). https://doi.org/10.1038/s41596-025-01166-4

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