SpringerLink
Log in

Scalable Generation of 3D Pancreatic Islet Organoids from Human Pluripotent Stem Cells in Suspension Bioreactors

  • Protocol
  • First Online:
Tissue Morphogenesis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2805))

Abstract

We describe a scalable method for the robust generation of 3D pancreatic islet-like organoids from human pluripotent stem cells using suspension bioreactors. Our protocol involves a 6-stage, 20-day directed differentiation process, resulting in the production of 104–105 organoids. These organoids comprise α- and β-like cells that exhibit glucose-responsive insulin and glucagon secretion. We detail methods for culturing, passaging, and cryopreserving stem cells as suspended clusters and for differentiating them through specific growth media and exogenous factors added in a stepwise manner. Additionally, we address quality control measures, troubleshooting strategies, and functional assays for research applications.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Institutional subscriptions

References

  1. Leavens KF et al (2022) Stem cell-based multi-tissue platforms to model human autoimmune diabetes. Mol Metab 66:101610

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Melton D (2021) The promise of stem cell-derived islet replacement therapy. Diabetologia 64(5):1030–1036

    Article  PubMed  PubMed Central  Google Scholar 

  3. Hogrebe NJ, Ishahak M, Millman JR (2023) Developments in stem cell-derived islet replacement therapy for treating type 1 diabetes. Cell Stem Cell 30(5):530–548

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Balboa D, Iworima DG, Kieffer TJ (2021) Human pluripotent stem cells to model islet defects in diabetes. Front Endocrinol (Lausanne) 12:642152

    Article  PubMed  Google Scholar 

  5. Sharon N et al (2019) A peninsular structure coordinates asynchronous differentiation with morphogenesis to generate pancreatic islets. Cell 176(4):790–804.e13

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Adams MT, Blum B (2022) Determinants and dynamics of pancreatic islet architecture. Islets 14(1):82–100

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Siehler J et al (2021) Engineering islets from stem cells for advanced therapies of diabetes. Nat Rev Drug Discov 20(12):920–940

    Article  CAS  PubMed  Google Scholar 

  8. Alvarez-Dominguez JR et al (2020) Circadian entrainment triggers maturation of human in vitro islets. Cell Stem Cell 26(1):108–122.e10

    Article  CAS  PubMed  Google Scholar 

  9. Pagliuca FW et al (2014) Generation of functional human pancreatic beta cells in vitro. Cell 159(2):428–439

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Velazco-Cruz L et al (2019) Acquisition of dynamic function in human stem cell-derived beta cells. Stem Cell Reports 12(2):351–365

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Veres A et al (2019) Charting cellular identity during human in vitro beta-cell differentiation. Nature 569(7756):368–373

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Osafune K et al (2008) Marked differences in differentiation propensity among human embryonic stem cell lines. Nat Biotechnol 26(3):313–315

    Article  CAS  PubMed  Google Scholar 

  13. Hogrebe NJ et al (2021) Generation of insulin-producing pancreatic beta cells from multiple human stem cell lines. Nat Protoc 16(9):4109–4143

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Barsby T et al (2022) Differentiating functional human islet-like aggregates from pluripotent stem cells. STAR Protoc 3(4):101711

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Sui L et al (2018) Beta-cell replacement in mice using human type 1 diabetes nuclear transfer embryonic stem cells. Diabetes 67(1):26–35

    Article  CAS  PubMed  Google Scholar 

  16. Leite NC et al (2020) Modeling type 1 diabetes in vitro using human pluripotent stem cells. Cell Rep 32(2):107894

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Sharon N et al (2019) Wnt signaling separates the progenitor and endocrine compartments during pancreas development. Cell Rep 27(8):2281–2291.e5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Rasouli N, Melton DA, Alvarez-Dominguez JR (2020) Purification of live stem-cell-derived islet lineage intermediates. Curr Protoc Stem Cell Biol 53(1):e111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Davis JC et al (2019) Live cell monitoring and enrichment of stem cell-derived beta cells using intracellular zinc content as a population marker. Curr Protoc Stem Cell Biol 51(1):e99

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Docherty FM et al (2021) ENTPD3 marks mature stem cell-derived beta-cells formed by self-aggregation in vitro. Diabetes 70(11):2554–2567

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Leavens KF et al (2020) Generation of a double insulin and somatostatin reporter line, SCSe001-A-3, for the advancement of stem cell-derived pancreatic islets. Stem Cell Res 50:102112

    Article  PubMed  PubMed Central  Google Scholar 

  22. Nair GG et al (2019) Recapitulating endocrine cell clustering in culture promotes maturation of human stem-cell-derived beta cells. Nat Cell Biol 21(2):263–274

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Parent AV et al (2022) Development of a scalable method to isolate subsets of stem cell-derived pancreatic islet cells. Stem Cell Reports 17(4):979–992

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Nair GG, Tzanakakis ES, Hebrok M (2020) Emerging routes to the generation of functional beta-cells for diabetes mellitus cell therapy. Nat Rev Endocrinol 16(9):506–518

    Article  PubMed  PubMed Central  Google Scholar 

  25. Vining KH, Mooney DJ (2017) Mechanical forces direct stem cell behaviour in development and regeneration. Nat Rev Mol Cell Biol 18(12):728–742

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Balboa D et al (2022) Functional, metabolic and transcriptional maturation of human pancreatic islets derived from stem cells. Nat Biotechnol 40(7):1042–1055

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Davis JC et al (2020) Glucose response by stem cell-derived beta cells in vitro is inhibited by a bottleneck in glycolysis. Cell Rep 31(6):107623

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Barsby T, Otonkoski T (2022) Maturation of beta cells: lessons from in vivo and in vitro models. Diabetologia 65(6):917–930

    Article  PubMed  PubMed Central  Google Scholar 

  29. Alvarez-Dominguez JR, Melton DA (2022) Cell maturation: hallmarks, triggers, and manipulation. Cell 185(2):235–249

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Wortham M, Sander M (2021) Transcriptional mechanisms of pancreatic beta-cell maturation and functional adaptation. Trends Endocrinol Metab 32(7):474–487

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Velazco-Cruz L, Goedegebuure MM, Millman JR (2020) Advances toward engineering functionally mature human pluripotent stem cell-derived beta cells. Front Bioeng Biotechnol 8:786

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This work was supported by grants from the NIH (K01DK129442 and DP1DK130673) and the Human Islet Research Network (U24DK104162) and by a pilot award from the Diabetes Research Center at the University of Pennsylvania (P30DK19525) to J.R.A-D.

Author information

Authors and Affiliations

  1. Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA

    Samuel D. Pollock, Israeli M. Galicia-Silva, Zoe L. Gruskin & Juan R. Alvarez-Dominguez

  2. Institute for Regenerative Medicine and Institute for Diabetes, Obesity and Metabolism, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA

    Samuel D. Pollock, Israeli M. Galicia-Silva, Mai Liu, Zoe L. Gruskin & Juan R. Alvarez-Dominguez

  3. Department of Bioengineering, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA

    Mai Liu

Authors
  1. Samuel D. Pollock
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Israeli M. Galicia-Silva
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mai Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zoe L. Gruskin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Juan R. Alvarez-Dominguez
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Juan R. Alvarez-Dominguez .

Editor information

Editors and Affiliations

  1. 303 Hoyt Laboratory, Princeton University, PRINCETON, NJ, USA

    Celeste M. Nelson

Rights and permissions

Reprints and permissions

Copyright information

© 2024 The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Pollock, S.D., Galicia-Silva, I.M., Liu, M., Gruskin, Z.L., Alvarez-Dominguez, J.R. (2024). Scalable Generation of 3D Pancreatic Islet Organoids from Human Pluripotent Stem Cells in Suspension Bioreactors. In: Nelson, C.M. (eds) Tissue Morphogenesis. Methods in Molecular Biology, vol 2805. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3854-5_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3854-5_4

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3853-8

  • Online ISBN: 978-1-0716-3854-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation