Effects of Different Perfusing Routes through The Portal Vein, Hepatic Vein, and Biliary Duct on Whole Rat Liver Decellularization

Document Type : Original Article


1 Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

2 Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran


Organ transplantation is the last therapeutic choice for end-stage liver failure, which is limited by the lack of
sufficient donors. Decellularized liver can be used as a suitable matrix for liver tissue engineering with clinical application
potential. Optimizing the decellularization procedure would obtain a biological matrix with completely removed cellular
components and preserved 3-dimensional structure. This study aimed to evaluate the decellularization efficacy through
three anatomical routes.

Materials and Methods:
In this experimental study, rat liver decellularization was performed through biliary duct (BD),
portal vein (PV), and hepatic vein (HV); using chemical detergents and enzymes. The decellularization efficacy was
evaluated by measurement of DNA content, extracellular matrix (ECM) total proteins, and glycosaminoglycans (GAGs).
ECM preservation was examined by histological and immunohistochemical (IHC) staining and scanning electron
microscopy (SEM). Scaffold biocompatibility was tested by the MTT assay for HepG2 and HUVEC cell lines.

Decellularization through HV and PV resulted in a transparent scaffold by complete cell removal, while the BD
route produced an opaque scaffold with incomplete decellularization. H&E staining confirmed these results. Maximum
DNA loss was obtained using 1% and 0.5% sodium dodecyl sulfate (SDS) in the PV and HV groups and the DNA
content decreased faster in the HV group. At the final stages, the proteins excreted in the HV and PV groups were
significantly less than the BD group. The GAGs level was diminished after decellularization, especially in the PV and
HV groups. In the HV and PV groups the collagen amount was significantly more than the BD group. The IHC and SEM
images showed that the ECM structure was preserved and cellular components were entirely removed. MTT assay
showed the biocompatibility of the decellularized scaffold.

The results revealed that the HV is a more suitable route for liver decellularization than the PV and BD.


Main Subjects

  1. Mazza G, Al-Akkad W, Rombouts K, Pinzani M. Liver tissue engineering: from implantable tissue to whole organ engineering. Hepatol Commun. 2018; 2(2): 131-141.
  2. Jadlowiec CC, Taner T. Liver transplantation: current status and challenges. World J Gastroenterol. 2016; 22(18): 4438-4445.
  3. Hillebrandt KH, Everwien H, Haep N, Keshi E, Pratschke J, Sauer IM. Strategies based on organ decellularization and recellularization. Transpl Int. 2019; 32(6): 571-585.
  4. Zhou P, Huang Y, Guo Y, Wang L, Ling C, Guo Q, et al. Decellularization and recellularization of rat livers with hepatocytes and endothelial progenitor cells. Artif Organs. 2016; 40(3): E25-E38.
  5. Wang X, Cui J, Zhang BQ, Zhang H, Bi Y, Kang Q, et al. Decellularized liver scaffolds effectively support the proliferation and differentiation of mouse fetal hepatic progenitors. J Biomed Mater Res A. 2014; 102(4): 1017-1025.
  6. Keane TJ, Swinehart IT, Badylak SF. Methods of tissue decellularization used for preparation of biologic scaffolds and in vivo relevance. Methods. 2015; 84: 25-34.
  7. Crapo PM, Gilbert TW, Badylak SF. An overview of tissue and whole organ decellularization processes. Biomaterials. 2011; 32(12): 3233-3243.
  8. Gilpin A, Yang Y. Decellularization strategies for regenerative medicine: from processing techniques to applications. Biomed Res Int. 2017; 2017: 9831534.
  9. Coronado RE, Somaraki-Cormier M, Natesan S, Christy RJ, Ong JL, Halff GA. Decellularization and solubilization of porcine liver for use as a substrate for porcine hepatocyte culture: method optimization and comparison. Cell Transplant. 2017; 26(12): 1840-1854.
  10. Park KM, Park SM, Yang SR, Hong SH, Woo HM. Preparation of immunogen-reduced and biocompatible extracellular matrices from porcine liver. J Biosci Bioeng. 2013; 115(2): 207-215.
  11. Hussein KH, Park KM, Kang KS, Woo HM. Biocompatibility evaluation of tissue engineered decellularized scaffolds for biomedical application. Mater Sci Eng C Mater Biol Appl. 2016; 67: 766-778.
  12. Abshagen K, Kuhla A, Genz B, Vollmar B. Anatomy and physiology of the hepatic circulation. In: Lanzer P, editor. PanVascular medicine. Berlin, Heidelberg: Springer Berlin Heidelberg; 2015: 3607-3629.
  13. Gilbert TW. Strategies for tissue and organ decellularization. J Cell Biochem. 2012; 113(7): 2217-2222.
  14. De Kock J, Ceelen L, De Spiegelaere W, Casteleyn C, Claes P, Vanhaecke T, et al. Simple and quick method for whole-liver decellularization: a novel in vitro three-dimensional bioengineering tool? Arch Toxicol. 2011; 85(6): 607-612.
  15. Farndale RW, Buttle DJ, Barrett AJ. Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim Biophys Acta. 1986; 883(2): 173-177.
  16. Reddy GK, Enwemeka CS. A simplified method for the analysis of hydroxyproline in biological tissues. Clin Biochem. 1996; 29(3): 225-229.
  17. Badylak SF, Taylor D, Uygun K. Whole-organ tissue engineering: decellularization and recellularization of three-dimensional matrix scaffolds. Annu Rev Biomed Eng. 2011; 13: 27-53.
  18. Gilbert TW, Sellaro TL, Badylak SF. Decellularization of tissues and organs. Biomaterials. 2006; 27(19): 3675-3683.
  19. Struecker B, Butter A, Hillebrandt K, Polenz D, Reutzel-Selke A, Tang P, et al. Improved rat liver decellularization by arterial perfusion under oscillating pressure conditions. J Tissue Eng Regen Med. 2017; 11(2): 531-541.
  20. Gilpin A, Yang Y. Decellularization strategies for regenerative medicine: from processing techniques to applications. Biomed Res Int. 2017; 2017: 9831534.
  21. Fazelian-Dehkordi K, Ardekani SFM, Talaei-Khozani T. Quality comparison of decellularized omentum prepared by different protocols for tissue engineering applications. Cell J. 2022; 24(5): 267-276.
  22. Gilbert TW, Freund JM, Badylak SF. Quantification of DNA in biologic scaffold materials. J Surg Res. 2009; 152(1): 135-139.
  23. Wang Y, Bao J, Wu Q, Zhou Y, Li Y, Wu X, et al. Method for perfusion decellularization of porcine whole liver and kidney for use as a scaffold for clinical-scale bioengineering engrafts. Xenotransplantation. 2015; 22(1): 48-61.
  24. Wang LR, Lin YQ, Wang JT, Pan LL, Huang KT, Wan L, et al. Recent advances in re-engineered liver: de-cellularization and re-cellularization techniques. Cytotherapy. 2015; 17(8): 1015-1024.
  25. Soto-Gutierrez A, Zhang L, Medberry C, Fukumitsu K, Faulk D, Jiang H, et al. A whole-organ regenerative medicine approach for liver replacement. Tissue Eng Part C Methods. 2011; 17(6): 677-686.
  26. Mazza G, Al-Akkad W, Telese A, Longato L, Urbani L, Robinson B, et al. Rapid production of human liver scaffolds for functional tissue engineering by high shear stress oscillation-decellularization. Sci Rep. 2017; 7(1): 5534.
  27. Sabetkish S, Kajbafzadeh AM, Sabetkish N, Khorramirouz R, Akbarzadeh A, Seyedian SL, et al. Whole-organ tissue engineering: decellularization and recellularization of three-dimensional matrix liver scaffolds. J Biomed Mater Res A. 2015; 103(4): 1498-1508.
  28. Eipel C, Abshagen K, Vollmar B. Regulation of hepatic blood flow: the hepatic arterial buffer response revisited. World J Gastroenterol. 2010; 16(48): 6046-6057.
  29. Lautt WW. Hepatic circulation: physiology and pathophysiology. San Rafael (CA): Morgan & Claypool Life Sciences; 2009.
  30. Treyer A, Musch A. Hepatocyte polarity. Compr Physiol. 2013; 3(1): 243-287.
  31. Taylor DA, Kren SM, Rhett K, Robertson MJ, Morrissey J, Rodriguez OE, et al. Characterization of perfusion decellularized whole animal body, isolated organs, and multi-organ systems for tissue engineering applications. Physiol Rep. 2021; 9(12): e14817.
  32. Wu Q, Bao J, Zhou YJ, Wang YJ, Du ZG, Shi YJ, et al. Optimizing perfusion-decellularization methods of porcine livers for clinicalscale whole-organ bioengineering. Biomed Res Int. 2015; 2015: 785474.
  33. Maghsoudlou P, Georgiades F, Smith H, Milan A, Shangaris P, Urbani L, et al. Optimization of liver decellularization maintains extracellular matrix micro-architecture and composition predisposing to effective cell seeding. PLoS One. 2016; 11(5): e0155324.
  34. Willemse J, Verstegen MMA, Vermeulen A, Schurink IJ, Roest HP, van der Laan LJW, et al. Fast, robust and effective decellularization of whole human livers using mild detergents and pressure controlled perfusion. Mater Sci Eng C Mater Biol Appl. 2020; 108: 110200.
  35. Gratzer PF, Harrison RD, Woods T. Matrix alteration and not residual sodium dodecyl sulfate cytotoxicity affects the cellular repopulation of a decellularized matrix. Tissue Eng. 2006; 12(10): 2975-2983.
  36. Yang W, Xia R, Zhang Y, Zhang H, Bai L. Decellularized liver scaffold for liver regeneration. Methods Mol Biol. 2018; 1577: 11-23.
  37. Hosseini V, Maroufi NF, Saghati S, Asadi N, Darabi M, Ahmad SNS, et al. Current progress in hepatic tissue regeneration by tissue engineering. J Transl Med. 2019; 17(1): 383.
  38. Arzumanian VA, Kiseleva OI, Poverennaya EV. The curious case of the HepG2 cell line: 40 years of expertise. Int J Mol Sci. 2021; 22(23): 13135.
  39. Cao Y, Gong Y, Liu L, Zhou Y, Fang X, Zhang C, et al. The use of human umbilical vein endothelial cells (HUVECs) as an in vitro model to assess the toxicity of nanoparticles to endothelium: a review. J Appl Toxicol. 2017; 37(12): 1359-1369.
  40. Panwar A, Das P, Tan LP. 3D hepatic organoid-based advancements in liver tissue engineering. Bioengineering (Basel). 2021; 8(11): 185.