Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Sustained perfusion of revascularized bioengineered livers heterotopically transplanted into immunosuppressed pigs

Nature Biomedical Engineering volume 4, pages 437–445 (2020)Cite this article

Subjects

An Author Correction to this article was published on 05 November 2019

This article has been updated

Abstract

Implanted bioengineered livers have not exceeded three days of continuous perfusion. Here we show that decellularized whole porcine livers revascularized with human umbilical vein endothelial cells and implanted heterotopically into immunosuppressed pigs whose spleens had been removed can sustain perfusion for up to 15 days. We identified peak glucose consumption rate as a main predictor of the patency of the revascularized bioengineered livers (rBELs). Heterotopic implantation of rBELs into pigs in the absence of anticoagulation therapy led to sustained perfusion for three days, followed by a pronounced immune responses directed against the human endothelial cells. A 10 day steroid-based immunosuppression protocol and a splenectomy at the time of rBEL implantation reduced the immune responses and resulted in continuous perfusion of the rBELs for over two weeks. We also show that the human endothelial cells in the perfused rBELs colonize the liver sinusoids and express sinusoidal endothelial markers similar to those in normal liver tissue. Revascularized liver scaffolds that can maintain blood perfusion at physiological pressures might eventually help to overcome the chronic shortage of transplantable human livers.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Porcine liver decellularization and perfusion bioreactor system.
Fig. 2: Analysis of rBEL culture kinetics and HUVEC phenotypic plasticity in decellularized liver matrix.
Fig. 3: In vitro and in vivo patency correlates with PGCR.
Fig. 4: Long-term in vivo perfusion studies in the presence and absence of immunosuppression.

Similar content being viewed by others

Data availability

The main data supporting the results in this study are available in the Article and Supplementary Information. The raw and analysed datasets generated during the study are available for research purposes from the corresponding authors on reasonable request.

Change history

References

  1. Shirakigawa, N., Takei, T. & Ijima, H. Base structure consisting of an endothelialized vascular-tree network and hepatocytes for whole liver engineering. J. Biosci. Bioeng. 116, 740–745 (2013).

    Article  CAS  PubMed  Google Scholar 

  2. Badylak, S. F., Taylor, D. & Uygun, K. Whole-organ tissue engineering: decellularization and recellularization of three-dimensional matrix scaffolds. Annu. Rev. Biomed. Eng. 13, 27–53 (2011).

    Article  CAS  PubMed  Google Scholar 

  3. Soto-Gutierrez, A. et al. A whole-organ regenerative medicine approach for liver replacement. Tissue Eng. Pt C 17, 677–686 (2011).

    Article  CAS  Google Scholar 

  4. Baptista, P. M. et al. The use of whole organ decellularization for the generation of a vascularized liver organoid. Hepatology 53, 604–617 (2011).

    Article  CAS  PubMed  Google Scholar 

  5. Barakat, O. et al. Use of decellularized porcine liver for engineering humanized liver organ. J. Surg. Res. 173, e11–e25 (2012).

    Article  CAS  PubMed  Google Scholar 

  6. Bao, J. et al. Construction of a portal implantable functional tissue-engineered liver using perfusion-decellularized matrix and hepatocytes in rats. Cell Transplant. 20, 753–766 (2011).

    Article  PubMed  Google Scholar 

  7. Zhou, P. et al. Decellularized liver matrix as a carrier for the transplantation of human fetal and primary hepatocytes in mice. Liver Transplant. 17, 418–427 (2011).

    Article  Google Scholar 

  8. Yagi, H. et al. Human-scale whole-organ bioengineering for liver transplantation: a regenerative medicine approach. Cell Transplant. 22, 231–242 (2013).

    Article  PubMed  Google Scholar 

  9. Uygun, B. E. et al. Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix. Nat. Med. 16, 814–820 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Bao, J. et al. Hemocompatibility improvement of perfusion-decellularized clinical-scale liver scaffold through heparin immobilization. Sci. Rep. 5, 10756 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Park, K. M. et al. Decellularized liver extracellular matrix as promising tools for transplantable bioengineered liver promotes hepatic lineage commitments of induced pluripotent stem cells. Tissue Eng. Pt A 22, 449–460 (2016).

    Article  CAS  Google Scholar 

  12. Wang, Y. et al. Recent advances in decellularization and recellularization for tissue-engineered liver grafts. Cells Tissues Organs 204, 125–136 (2017).

    Article  PubMed  Google Scholar 

  13. Uygun, B. E. & Yarmush, M. L. Engineered liver for transplantation. Curr. Opin. Biotechnol. 24, 893–899 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Mirmalek-Sani, S.-H., Sullivan, D. C., Zimmerman, C., Shupe, T. D. & Petersen, B. E. Immunogenicity of decellularized porcine liver for bioengineered hepatic tissue. Am. J. Pathol. 183, 558–565 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Uzarski, J. S. et al. Dual-purpose bioreactors to monitor noninvasive physical and biochemical markers of kidney and liver scaffold recellularization. Tissue Eng. Pt C 21, 1032–1043 (2015).

    Article  CAS  Google Scholar 

  16. Robertson, M. J., Soibam, B., O’Leary, J. G., Sampaio, L. C. & Taylor, D. A. Recellularization of rat liver: an in vitro model for assessing human drug metabolism and liver biology. PLoS ONE 13, e0191892 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Mazza, G., Al-Akkad, W., Rombouts, K. & Pinzani, M. Liver tissue engineering: from implantable tissue to whole organ engineering. Hepatol. Commun. 2, 131–141 (2018).

    Article  PubMed  Google Scholar 

  18. Rogers, S. C., Zhang, X., Azhar, G., Luo, S. & Wei, J. Y. Exposure to high or low glucose levels accelerates the appearance of markers of endothelial cell senescence and induces dysregulation of nitric oxide synthase. J. Gerontol. A 68, 1469–1481 (2013).

    Article  CAS  Google Scholar 

  19. Lalor, P. F., Lai, W. K., Curbishley, S. M., Shetty, S. & Adams, D. H. Human hepatic sinusoidal endothelial cells can be distinguished by expression of phenotypic markers related to their specialised functions in vivo. World J. Gastroenterol. 12, 5429–5439 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. MacParland, S. A. et al. Single cell RNA sequencing of human liver reveals distinct intrahepatic macrophage populations. Nat. Commun. 9, 4383 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  21. DeLeve, L. D. Liver sinusoidal endothelial cells in hepatic fibrosis. Hepatology 61, 1740–1746 (2015).

    Article  CAS  PubMed  Google Scholar 

  22. Skovseth, D. K., Yamanaka, T., Brandtzaeg, P., Butcher, E. C. & Haraldsen, G. Vascular morphogenesis and differentiation after adoptive transfer of human endothelial cells to immunodeficient mice. Am. J. Pathol. 160, 1629–1637 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Leach, L., Hamilton, R. D. & Foss, A. J. E. Phenotypic plasticity of human umbilical vein endothelial cells. Br. J. Ophthalmol. 96, 1152 (2012).

    Article  PubMed  Google Scholar 

  24. Robb, R. A. The biomedical imaging resource at Mayo Clinic. IEEE Trans. Med. Imaging 20, 854–867 (2001).

    Article  CAS  PubMed  Google Scholar 

  25. Nyberg, S. L., Amiot, B., Hardin, J., Baskin-Bey, E. & Platt, J. L. Cytotoxic immune response to a xenogeneic bioartificial liver. Cell Transplant. 13, 783–792 (2004).

    Article  PubMed  Google Scholar 

  26. Kin, W. R. et al. OPTN/SRTR 2016 annual data report: liver. Am. J. Transplant. 18, 172–253 (2018).

    Article  Google Scholar 

  27. Lopez, P. M. & Martin, P. Update on liver transplantation: indications, organ allocation, and long-term care. Mt Sinai J. Med. 73, 1056–1066 (2006).

    PubMed  Google Scholar 

  28. Dhawan, A., Puppi, J., Hughes, R. D. & Mitry, R. R. Human hepatocyte transplantation: current experience and future challenges. Nat. Rev. Gastroenterol. Hepatol. 7, 288–298 (2010).

    Article  PubMed  Google Scholar 

  29. Suchy, F., Yamaguchi, T. & Nakauchi, H. iPSC-derived organs in vivo: challenges and promise. Cell Stem Cell 22, 21–24 (2018).

    Article  CAS  PubMed  Google Scholar 

  30. Nicolas, C., Wang, Y. & Nyberg, S. L. Cell therapy in chronic liver disease. Curr. Opin. Gastroenterol. 32, 189–194 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Hussein, K. H., Park, K. M., Kang, K. S. & Woo, H. M. Heparin-gelatin mixture improves vascular reconstruction efficiency and hepatic function in bioengineered livers. Acta Biomater. 38, 82–93 (2016).

    Article  CAS  PubMed  Google Scholar 

  32. Ko, I. K. et al. Bioengineered transplantable porcine livers with re-endothelialized vasculature. Biomaterials 40, 72–79 (2015).

    Article  CAS  PubMed  Google Scholar 

  33. Mei, J. et al. The angiogenesis in decellularized scaffold-mediated the renal regeneration. Oncotarget 7, 27085–27093 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Guan, Y. et al. The effective bioengineering method of implantation decellularized renal extracellular matrix scaffolds. Oncotarget 6, 36126–36138 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Doi, R. et al. Transplantation of bioengineered rat lungs recellularized with endothelial and adipose-derived stromal cells. Sci. Rep. 7, 8447 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Mao, S., Glorioso, J., Elgilani, F., De Lorenzo, S. & Deeds, M. Sustained in vivo perfusion of a re-endothelialized tissue engineered porcine liver. Int. J. Transplant. Res. Med. 3, 031 (2017).

    Google Scholar 

  37. Dudakovic, A. et al. High-resolution molecular validation of self-renewal and spontaneous differentiation in clinical-grade adipose-tissue derived human mesenchymal stem cells. J. Cell. Biochem. 115, 1816–1828 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Kalari, K. R. et al. MAP-RSeq: Mayo analysis pipeline for RNA sequencing. BMC Bioinformatics 15, 224 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013).

    PubMed  PubMed Central  Google Scholar 

  40. Anders, S., Pyl, P. T. & Huber, W. HTSeq–a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015).

    CAS  PubMed  Google Scholar 

  41. Metsalu, T. & Vilo, J. ClustVis: a web tool for visualizing clustering of multivariate data using Principal Component Analysis and heatmap. Nucleic Acids Res. 43, W566–W570 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Huang, D. W., Sherman, B. T. & Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank C. Verfaillie and M. Kumar from the Stem Cell Institute Leuven, KU Leuven, Belgium for the qPCR analysis performed in the manuscript, the Mayo Clinic Microscopy and Cell Analysis Core for experimental and technical support and the Mayo Clinic Biomedical Imaging Resource for creating the 3D visualizations from CT images. We also thank L. Wentz for assistance with cell culture and J. Uzarski and M. M. Macenski for their review of and comments on the final manuscript. This work was made possible by financial support from Miromatrix, Mayo Clinic ILP grants and the National Institutes of Health (grant no. R01DK106667 to S.L.N.; grant nos. R01DK117861 and R03DK113339 to R.C.H.).

Author information

Author notes

  1. These authors contributed equally: Mohammed F. Shaheen, DongJin Joo.

Authors and Affiliations

  1. William J. von Liebig Center for Transplantation and Clinical Regeneration, Mayo Clinic, Rochester, MN, USA

    Mohammed F. Shaheen, Dong Jin Joo, Harvey S. Chen, Yi Li, Bruce Amiot, Erek Nelson & Scott L. Nyberg

  2. Department of Surgery, Mayo Clinic, Rochester, MN, USA

    Mohammed F. Shaheen, Harvey S. Chen, Erek Nelson & Scott L. Nyberg

  3. Department of Surgery, Yonsei University College of Medicine, Seoul, South Korea

    Dong Jin Joo

  4. Miromatrix Medical Inc., Eden Prairie, MN, USA

    Jeffrey J. Ross, Brett D. Anderson, Anne Young, Viviana Zlochiver, Benjamin G. Steiner & Dominique Seetapun Davidow

  5. Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA

    Robert C. Huebert, Vijay H. Shah & Mengfei Liu

  6. Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA

    Taofic Mounajjed & Allan B. Dietz

  7. Department of Radiology, Mayo Clinic, Rochester, MN, USA

    Gregory Michalak

  8. Center for Regenerative Medicine, Mayo Clinic, Rochester, MN, USA

    Christopher R. Paradise

  9. Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA

    Andre J. van Wijnen

  10. Department of Orthopedics, Mayo Clinic, Rochester, MN, USA

    Andre J. van Wijnen

Authors
  1. Mohammed F. Shaheen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dong Jin Joo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jeffrey J. Ross
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Brett D. Anderson
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Harvey S. Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Robert C. Huebert
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yi Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Bruce Amiot
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Anne Young
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Viviana Zlochiver
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Erek Nelson
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Taofic Mounajjed
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Allan B. Dietz
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Gregory Michalak
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Benjamin G. Steiner
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Dominique Seetapun Davidow
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Christopher R. Paradise
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Andre J. van Wijnen
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Vijay H. Shah
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Mengfei Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Scott L. Nyberg
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.F.S., J.J.R., D.S.D. and S.L.N. designed the study. A.Y. and V.Z. performed the HUVEC and rBEL culture. M.F.S., D.J.J., H.S.C., Y.L., B.A., E.N., T.M. and S.L.N. performed the surgical procedures. G.M. performed the CT. B.A., R.C.H. and M.L. performed the electron microscopy, B.G.S. performed the light and fluorescence microscopy. A.J.v.W. and C.R.P. performed the RNA-seq analysis. M.F.S., D.J.J., J.J.R., B.D.A., D.S.D., R.C.H., V.H.S., M.L. and S.L.N. analysed the experimental data. B.D.A. drafted the figures. M.F.S., J.J.R., B.D.A. and S.L.N. wrote the manuscript. R.C.H., V.H.S., A.B.D. and M.L. reviewed and edited the manuscript.

Corresponding authors

Correspondence to Jeffrey J. Ross or Scott L. Nyberg.

Ethics declarations

Competing interests

Miromatrix Medical Inc. is a privately funded company and owns the patent rights for the perfusion decellularization and recellularization technologies employed in this study. The research was funded by Miromatrix and a Mayo Clinic Innovation grant. J.J.R., B.D.A., A.Y., B.G.S. and D.S.D. are employees of Miromatrix.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Supplementary Information

Supplementary figures and tables.

Reporting Summary

Supplementary Dataset 1

RNA-seq DAVID analysis.

Supplementary Dataset 2

Upregulated genes.

Supplementary Dataset 3

Similarity matrix input genes.

Supplementary Video 1

Intraoperative footage of the perfusion of the BEL graft.

Supplementary Video 2

3D CT reconstruction animation depicting the postoperative anatomy of the rBEL.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Shaheen, M.F., Joo, D.J., Ross, J.J. et al. Sustained perfusion of revascularized bioengineered livers heterotopically transplanted into immunosuppressed pigs. Nat Biomed Eng 4, 437–445 (2020). https://doi.org/10.1038/s41551-019-0460-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41551-019-0460-x

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research