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Perfusion decellularization of whole organs

Nature Protocols volume 9, pages 1451–1468 (2014)Cite this article

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Abstract

The native extracellular matrix (ECM) outlines the architecture of organs and tissues. It provides a unique niche of composition and form, which serves as a foundational scaffold that supports organ-specific cell types and enables normal organ function. Here we describe a standard process for pressure-controlled perfusion decellularization of whole organs for generating acellular 3D scaffolds with preserved ECM protein content, architecture and perfusable vascular conduits. By applying antegrade perfusion of detergents and subsequent washes to arterial vasculature at low physiological pressures, successful decellularization of complex organs (i.e., hearts, lungs and kidneys) can be performed. By using appropriate modifications, pressure-controlled perfusion decellularization can be achieved in small-animal experimental models (rat organs, 4–5 d) and scaled to clinically relevant models (porcine and human organs, 12–14 d). Combining the unique structural and biochemical properties of native acellular scaffolds with subsequent recellularization techniques offers a novel platform for organ engineering and regeneration, for experimentation ex vivo and potential clinical application in vivo.

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Figure 1: Layout of small-organ decellularization setup.
Figure 2: Overview of large-organ decellularization setup.
Figure 3: Cannulation of large organs.
Figure 4: Time-lapse images of the SDS phase of the decellularization process.
Figure 5: Validation of decellularization process.
Figure 6: Immunohistological and biochemical characterization of organs post decellularization.

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Acknowledgements

The present study was supported by the US National Institutes of Health (NIH) Director's New Innovator Award DP2 OD008749-01, grants R21 HL108663-01 and R01 HL108678, by seed grants by the Department of Surgery, Massachusetts General Hospital and the Harvard Stem Cell Institute, and a research grant by United Therapeutics Inc. We thank D.J. Mathisen and J.P. Vacanti for their mentorship and senior advice on surgical procedures, tissue engineering aspects and for critical review of the manuscript.

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Author notes

  1. Jacques P Guyette and Sarah E Gilpin: These authors contributed equally to this work.

Authors and Affiliations

  1. Center for Regenerative Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA

    Jacques P Guyette, Sarah E Gilpin, Jonathan M Charest, Xi Ren & Harald C Ott

  2. Harvard Medical School, Boston, Massachusetts, USA

    Jacques P Guyette, Sarah E Gilpin, Luis F Tapias, Xi Ren & Harald C Ott

  3. Department of Surgery, Division of Thoracic Surgery, Massachusetts General Hospital, Boston, Massachusetts, USA

    Luis F Tapias & Harald C Ott

  4. Harvard Stem Cell Institute, Boston, Massachusetts, USA

    Harald C Ott

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  1. Jacques P Guyette
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Contributions

H.C.O. conceived and designed the initial protocol, and oversaw data collection and writing of the manuscript. J.P.G. provided further development of heart and kidney decellularization protocols, oversaw data analysis and figure creation, and contributed to the manuscript and review. S.E.G. provided further development of lung and kidney decellularization protocols, oversaw data analysis and figure creation, and contributed to the manuscript and review. J.M.C. provided biochemical and histological data characterization, oversaw figure creation, and contributed to the manuscript. L.F.T. provided biochemical and histological data characterization, and contributed to manuscript and revisions. X.R. provided characterization of decellularized organ vasculature.

Corresponding author

Correspondence to Harald C Ott.

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Competing interests

H.C.O. is the founder and stockholder of IVIVA Medical, Inc. This relationship did not affect the content or conclusions contained in this manuscript.

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Supplementary Methods

Immunohistochemistry and biochemical analysis. (PDF 106 kb)

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Guyette, J., Gilpin, S., Charest, J. et al. Perfusion decellularization of whole organs. Nat Protoc 9, 1451–1468 (2014). https://doi.org/10.1038/nprot.2014.097

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