Use of decellularized porcine liver for engineering humanized liver organ.

BACKGROUND New bioartificial liver devices are needed to supplement the limited supply of organ donors available for patients with end-stage liver disease. Here, we report the results of a pilot study aimed at developing a humanized porcine liver by transplanting second trimester human fetal hepatocytes (Hfh) co-cultured with fetal stellate cells (Hfsc) into the decellularized matrix of a porcine liver. MATERIAL AND METHODS Ischemic livers were removed from 19 Yorkshire swine. Liver decellularization was achieved by an anionic detergent (SDS). The decellularized matrix of three separate porcine liver matrices was seeded with 3.5 × 10(8) and 1 × 10(9) of Hfsc and Hfh, respectively, and perfused for 3, 7, and 13 d. The metabolic and synthetic activities of the engrafted cells were assessed during and after perfusion. RESULTS Immunohistologic examination of the decellularized matrix showed removal of nuclear materials with intact architecture and preserved extracellular matrix (ECM) proteins. During perfusion of the recellularized matrices, measurement of metabolic parameters (i.e., oxygen concentration, glucose consumption, and lactate and urea production) indicated active metabolism. The average human albumin concentration was 29.48 ± 7.4 μg/mL. Immunohistochemical analysis revealed cell differentiation into mature hepatocytes. Moreover, 40% of the engrafted cells were actively proliferating, and less than 30% of cells were apoptotic. CONCLUSION We showed that our decellularization protocol successfully removed the cellular components of porcine livers while preserving the native architecture and most ECM protein. We also demonstrated the ability of the decellularized matrix to support and induce phenotypic maturation of engrafted Hfh in a continuously perfused system.

[1]  S. Koenig,et al.  Maintaining hepatocyte differentiation in vitro through co-culture with hepatic stellate cells , 2009, In Vitro Cellular & Developmental Biology - Animal.

[2]  李亚明,et al.  Hepatocyte transplantation , 2005 .

[3]  Anna-Karin Sohlenius-Sternbeck,et al.  Determination of the hepatocellularity number for human, dog, rabbit, rat and mouse livers from protein concentration measurements , 2006 .

[4]  M. Najimi,et al.  Cell transplantation in the treatment of liver diseases , 2008, Pediatric transplantation.

[5]  宇山 直樹.  Regulation of cultured rat hepatocyte proliferation by stellate cells , 2002 .

[6]  J. Chowdhury,et al.  Hepatocyte transplantation , 2004, American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons.

[7]  J. Lehmann,et al.  Cultivation of immortalized human hepatocytes HepZ on macroporous CultiSpher G microcarriers. , 2000, Biotechnology and bioengineering.

[8]  J. Gerlach,et al.  Bioartificial liver systems: why, what, whither? , 2008, Regenerative medicine.

[9]  K. Shakesheff,et al.  Liver tissue engineering: a role for co-culture systems in modifying hepatocyte function and viability. , 2001, Tissue engineering.

[10]  P. Lewindon,et al.  The role of hepatic stellate cells and transforming growth factor-beta(1) in cystic fibrosis liver disease. , 2002, The American journal of pathology.

[11]  W. Marks,et al.  Organ donation and utilization in the United States, 2004 , 2005, American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons.

[12]  Stephen F Badylak,et al.  Xenogeneic extracellular matrix as a scaffold for tissue reconstruction. , 2004, Transplant immunology.

[13]  Stephen F Badylak,et al.  Quantification of DNA in biologic scaffold materials. , 2009, The Journal of surgical research.

[14]  Alejandro Soto-Gutiérrez,et al.  Differentiation and transplantation of human embryonic stem cell-derived hepatocytes. , 2009, Gastroenterology.

[15]  J. Yager,et al.  A morphological study of differentiated hepatocytes in vitro , 1995, Hepatology.

[16]  A. Bader,et al.  Bioreactor developments for tissue engineering applications by the example of the bioartificial liver. , 2002, Advances in biochemical engineering/biotechnology.

[17]  Vera Rogiers,et al.  Molecular mechanisms underlying the dedifferentiation process of isolated hepatocytes and their cultures. , 2006, Current drug metabolism.

[18]  Thomas M van Gulik,et al.  Functional and morphological comparison of three primary liver cell types cultured in the AMC bioartificial liver , 2007, Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society.

[19]  Kevin M. Shakesheff,et al.  The Effect of Three-Dimensional Co-Culture of Hepatocytes and Hepatic Stellate Cells on Key Hepatocyte Functions in vitro , 2005, Cells Tissues Organs.

[20]  J. Punch,et al.  Organ Donation and Utilization in the United States, 1996–2005 , 2007, American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons.

[21]  S. Badylak,et al.  Extracellular matrix as a biological scaffold material: Structure and function. , 2009, Acta biomaterialia.

[22]  K. Cherian,et al.  Technique to process xenogenic tissues for cardiovascular implantation - : A preliminary report , 2006 .

[23]  D J Mooney,et al.  Dynamic seeding and in vitro culture of hepatocytes in a flow perfusion system. , 2000, Tissue engineering.

[24]  Doris A Taylor,et al.  Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart , 2008, Nature Medicine.

[25]  M L Yarmush,et al.  Effect of cell–cell interactions in preservation of cellular phenotype: cocultivation of hepatocytes and nonparenchymal cells , 1999, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[26]  N. Futran,et al.  Crosslinking of an oesophagus acellular matrix tissue scaffold , 2008, Journal of tissue engineering and regenerative medicine.

[27]  Robert L. Thompson,et al.  Hepatic maturation of human fetal hepatocytes in four-compartment three-dimensional perfusion culture. , 2010, Tissue engineering. Part C, Methods.

[28]  R. Fisher,et al.  Human hepatocyte transplantation: worldwide results. , 2006, Transplantation.

[29]  J. Gerlach,et al.  Human fetal hepatocyte behavior in dynamic 3D perfusion culture bioreactors , 2007 .

[30]  J. Lemire,et al.  Cell lineages and oval cell progenitors in rat liver development. , 1991, Cancer research.

[31]  Mitsuru Sato,et al.  Hepatic stellate cells: unique characteristics in cell biology and phenotype. , 2003, Cell structure and function.

[32]  S. Seki,et al.  Regulation of cultured rat hepatocyte proliferation by stellate cells. , 2002, Journal of hepatology.

[33]  N. Sussman,et al.  Artificial liver: A forthcoming attraction , 1993, Hepatology.

[34]  Stephen F Badylak,et al.  The extracellular matrix as a biologic scaffold material. , 2007, Biomaterials.

[35]  N. Fausto,et al.  Establishment, characterization, and long‐term maintenance of cultures of human fetal hepatocytes , 2003, Hepatology.

[36]  J. Seppen,et al.  In Vitro Functionality of Human Fetal Liver Cells and Clonal Derivatives under Proliferative Conditions , 2006, Cell transplantation.

[37]  Hiroshi Yagi,et al.  Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix , 2010, Nature Medicine.

[38]  Anna-Karin Sohlenius-Sternbeck,et al.  Determination of the hepatocellularity number for human, dog, rabbit, rat and mouse livers from protein concentration measurements. , 2007, Toxicology in vitro : an international journal published in association with BIBRA.

[39]  Masayuki Yamato,et al.  Engineering functional two- and three-dimensional liver systems in vivo using hepatic tissue sheets , 2007, Nature Medicine.