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Sustained perfusion of revascularized bioengineered livers heterotopically transplanted into immunosuppressed pigs

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

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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.

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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.

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.

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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.).

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Authors

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.

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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.

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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

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