New Method of Hepatocyte Transplantation and Extracorporeal Liver Support

Abstract

A technique has been developed by the authors that allows hepatocyte attachment on collagen-coated microcarriers resulting in prolonged hepatocyte viability and function both in vivo and in vitro. Rat hepatocytes were obtained by portal vein collagenase perfusion. Intraperitoneally transplanted microcarrier-attached normal hepatocytes into congeneic Gunn rats were functioning 3-4 weeks later, as shown by the presence and persistence of conjugated bilirubin in recipient bile, sustained decrease in serum bilirubin, uptake of Tc99m-DESIDA, and morphologic criteria. Intraperitoneal transplantation of normal microcarrier-attached hepatocytes into genetically albumin deficient rats (NAR) resulted in marked increase in plasma albumin levels (6 days without and 21 days with Cyclosporin A immunosuppression). Microcarrier-attached hepatocytes transplanted after 2 weeks of storage at -80 C into congeneic Gunn rats were viable and functional as assessed by criteria outlined above. An extracorporeal liver perfusion system was developed using the microcarrier-attached hepatocytes that was capable of synthesizing and conjugating bilirubin and synthesizing liver-specific proteins.

Survival, organization, and function of microcarrier-attached hepatocytes transplanted in rats.

“…As reported elsewhere, i.p. injection of hepatocytes in Gunn rats without attachment to the microcarriers did not result in the excretion of conjugated bilirubin in bile (12). Prolonged survival and function of hepatocytes observed in this study suggests that attachment of hepatocytes to the collagen-coated surface of the microcarriers may improve survival and differentiated function of the transplanted hepatocytes by providing a matrix for cell attachment.…”

“…Bile was collected from recipient Gunn rats through bile duct fistulae, and bile pigments were analyzed by reverse-phase HPLC (18) as described elsewhere (12). For characterization of the conjugated bilirubin excreted in bile, aliquots of the bile were treated with bacterial B-glucuronidase (12) before bile pigment analysis (19,20).…”

Current development of bioreactors for extracorporeal bioartificial liver (Review)

“…142 HepatAssist was extensively evaluated by phase I clinical trial. The clinical research achievement of HepatAssits was seen as a milestone in the area of EBAL R&D. The bioreactor of HepatAssit is based on the hollow fiber configuration with ͑5-7͒ ϫ 10 9 cryopreserved primary porcine hepatocytes attached on microcarrier culturing in its extracapillary space.…”

Intracellular nucleotide pools and ratios as tools for monitoring dedifferentiation of primary porcine hepatocytes in culture

“…Cell adhesion to matrix scaffolds is necessary for cells to metabolize, survive or proliferate (Boudreau et al 1995;Fang et al 1996;Frisch and Francis 1994;Meredith et al 1993;Re et al 1994;Zhu et al 1996). A large number of liver cell culture techniques have been developed to promote cell organization with the aim of providing conditions similar to in vivo conditions Cultivation on microcarriers (Demetriou et al 1986), in the extra-capillary space of a multibundle hollow fibre membrane network (Gerlach et al 1994), within a non-woven fabric mesh (Flendrig et al 1997), co-culture with non-parenchymal cells in an extracellular matrix environment (Bucher et al 1990;Bader et al 1995;Yagi et al 1998;Tilles et al 2001), cultivation of primary hepatocytes in spheroids (Tobe et al 1992;Kobayashi et al 1994) or use of the so-called sandwich model (Dunn et al 1989; have been reported to improve organization similar to the micro-architecture of hepatocyte monolayers found in vivo and of extending the presence of differentiation markers.…”