High-Yield Preparation of Porcine Hepatocytes for Long Survival after Transplantation in the Spleen

Abstract

The creation of an auxiliary liver by autotransplantation of liver parenchymal cells into the spleen has mainly been studied in rats for the treatment of acute liver failure. In order to apply this procedure to humans with chronic liver insufficiency the aim of this work was: (1) To demonstrate that hepatocytes can survive for long periods after autotransplantation into the spleen; (2) to increase the yield of the isolation of hepatocytes obtained from pig livers since this animal has a more fibrous liver than rats or normal humans and consequently one which is more difficult to dissociate. In 21 pigs isolated hepatocytes were obtained with in collagenase dissociation technique, the yield being 1–3 × 10⁷ cells per gram of liver and the viability 70–95%. The hepatocytes survived and maintained normal morphological and histochemical characteristics up to 7 months after transplantation, the date of sacrifice of the last animal.

Mouse hepatocytes migrate to liver parenchyma and function indefinitely after intrasplenic transplantation.

“…Although several previous studies (reviewed in refs. 1 and 2) have used histological techniques to demonstrate hepatocyte-like cells within the spleen (3)(4)(5)(6)(7)(8)(9)(10)(11)(12), fat pads (13), pancreas (14,15), or subrenal capsule (16), or on microcarrier beads in the peritoneum (17), after hepatocyte injections, the number of surviving cells and their degree of liver function have been impossible to assess due to the absence of quantitative biochemical markers and/or rejection of transplanted cells. Short-term (4,(17)(18)(19)(20) or long-term (3,14) reconstitution of some hepatic function has been demonstrated by transplanting normal hepatocytes into rats with a genetic deficiency of uridine diphosphate (UDP)-glucuronyltransferase, but it is unclear how many surviving hepatocytes were required to produce the slight decrease in serum bilirubin and increase in conjugated bilirubin in the bile.…”

Hepatocyte transplantation: Back to the future

“…Kusano and Mito (20) initially reported indefinite survival of syngeneic hepatocytes transplanted into the splenic pulp. Several groups have confirmed these observations (21)(22)(23)(24). Hepatocyte survival in other ectopic sites has been varied.…”

Hepatocyte transplantation

“…Further separation of liver cells can be achieved with elutriation techniques. 6 In larger animals, problems have related to obtaining sufficient yield and the high cost of materials. Many investigators use recirculation methods so that enzymes used to digest liver tissue can be used.…”

“…Many investigators use recirculation methods so that enzymes used to digest liver tissue can be used. 6 A number of different sites have been used for hepatocyte tran~plantation.~-~~ Site consideration includes immediate vascular circulation to the transplanted cells, optimal provision of growth condit i o n~,~~ protection from the immune system of the host, and ease of monitoring.14 Transplant sites have included splenic pulp, serial subcapsular space, portal vein, intramesenteric, pancreas, lungs, intracapsular fat pad, and peritoneal ~a v i t y .~-~~ The splenic architecture has been reported to be particularly well suited to promoting growth of hepatocytes in small animal models such as the rat. The reticular framework of the spleen has been reported to play an important role in supporting hepatocyte growth first as a trap for injected cells and secondarily by the interaction of the reticulum and proliferating hepato~y t e s .~J '~~* The success of hepatocyte transplantation into the spleen may in part be explained by the observation that a substantial number of hepatocytes placed within the spleen traverse the splenic vein into the portal circulation and end up in the liver where they can be long 1 i~e d .…”