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A new high-water-content (83%) and highly permeable anionic polyelectrolyte hydrogel was obtained by phase inversion of a polymer solution containing 6% polyacrylonitrile-sodium methallylsulphonate, 91% dimethylsulphoxide and 3% physiological saline solution. Hydrogel-based hollow fibres (HFs) were fabricated with a co-extrusion apparatus in collaboration with Hospal (France). HFs have an internal diameter of 800 microns and a wall thickness of 100 microns. Experimental results demonstrated that hydrogel-based HFs were permeable to albumin (mol. wt 69,000) and human immunoglobulin G (150,000), but were impermeable to immunoglobulins A (170,000) and M (900,000) after 24 h of diffusion. In vitro, the viability of isolated rat hepatocytes injected into the HFs was 64 +/- 6% after 10 d versus 30 +/- 5% for hepatocytes cultured in Petri dishes (P = 0.0001). Under these conditions, the amount of albumin released by encapsulated hepatocytes was 12 +/- 3 micrograms/24 h/10(6) cells at day 10, whereas at that time no albumin was released by hepatocytes cultured in Petri dishes. In vivo, histological study of hydrogel HFs implanted up to 6 wk in the peritoneum of rats revealed a low inflammatory tissue reaction without giant multinucleate cells in the foreign tissue, which decreased after the third week. The survival rate of encapsulated hepatocytes was over 85% 45 d after transplantation in the peritoneum of syngeneic Lewis rats. Therefore, this hydrogel demonstrates highly favourable properties for encapsulation of hepatocytes with regard to its biocompatibility, permeability and ability to maintain hepatocytes in a functional state for prolonged periods.

Hepatocyte transplantation could be an alternative to whole liver transplantation. Allogeneic hepatocytes are rejected if transplanted without immunosuppression. The aim of this study was to transplant allogeneic hepatocytes in the peritoneum and to protect them from rejection by encapsulation in a new semipermeable membrane.Rat hepatocytes were encapsulated in hydrogel-based hollow fibers, obtained from AN69 copolymer, before being transplanted into the peritoneum of rats. Outcome of allogeneic hepatocytes encapsulated in hollow fibers was compared with that of syngeneic hepatocytes encapsulated in hollow fibers, with that of free allogeneic hepatocytes, and with allogeneic hepatocytes encapsulated in hollow fibers left open. Cell viability was assessed by erythrosin exclusion, structure by electron microscopy, and function by albumin release.Up to 90 days, viability of allogeneic hepatocytes in hollow fibers was greater than 80%. The structure remained normal at electron microscopy. Albumin release was 16.5 +/- 0.3 micrograms/24 hr/10(6) hepatocytes (day 15), 14.2 +/- 2.0 micrograms/24 hr/10(6) hepatocytes (day 30), 8.8 +/- 0.1 micrograms/24 hr/10(6) hepatocytes (day 60), and 11.4 +/- 0.3 micrograms/24 hr/10(6) hepatocytes (day 90). Free hepatocytes and hepatocytes in hollow fibers left open did not survive at day 15.Viability and function of encapsulated allogeneic hepatocytes were maintained up to 90 days after transplantation, without immunosuppression.

The liver is the most frequent site of metastases in colon cancer. No good animal model has been available to help improve the treatment of liver metastases or their prevention after resection of a primary colon cancer. The aim of this study was to develop a model of colon cancer induced by azoxymethane in the rat and to study the outcome after surgical resection alone or in association with intraperitoneal chemotherapy (5-fluorouracil (5-FU). Three hundred male Wistar rats received subcutaneous azoxymethane (10 mg/kg body weight/week) for 12 weeks. Eighty-three rats with isolated colon cancer underwent total colectomy; 40 of these rats with no metastases were randomized into two groups: surgery alone or surgery plus 5-FU (5 mg/kg body weight/day) for 5 days after surgery. Thirty rats were able to be evaluated. At autopsy, peritoneal carcinomatosis and liver metastases were more frequent in the control group than after adjuvant treatment with 5-FU (27.7 percent vs. 0, P less than 0.05; and 22.2 percent vs. 0, P less than 0.05, respectively). The rates of peritoneal and hepatic recurrence after resection of the primary cancer indicate that the model mimics the natural history of human colon cancer. In this model, 5-FU reduced the rate of peritoneal carcinomatosis and liver metastases but did not influence survival.

To determine the viability and differentiation of human hepatocytes immunoprotected by encapsulation and transplanted in rats without immunosuppression.Freshly isolated human hepatocytes were encapsulated in hollow fibers and transplanted in the peritoneal cavity of immunocompetent rats. The fibers were explanted for analysis at D3, D7 and D14 following transplantation. Morphological features under light and electron microscopy and gene expression were compared to those of non-transplanted encapsulated hepatocytes (D0). Human cytochrome P450 3A and albumin mRNAs were quantified by Northern blot. Cytochrome P450 3A proteins were detected by Western blot and cytochrome P450 3A enzyme activity was assessed by measuring the formation of 6beta-hydroxytestosterone by high performance liquid chromatography.Transplanted hepatocytes were more than 60 % viable and exhibited morphological criteria of hepatocytic differentiation up to D7. Albumin and cytochrome P450 3A transcripts were also detected up to D14. At D3 and D7, albumin mRNA levels were of 30 %, compared to control D0 hepatocytes, while cytochrome P450 3A5 and cytochrome P450 3A4 mRNA levels were 65 % and 0 %, respectively. Cytochrome P450 3A immunoreactivity was detected by Western blot up to D14 and 6beta-hydroxylase activity was 17 % at D3 compared to D0, supporting with disappearance of cytochrome P450 3A4 mRNA.Human hepatocytes remain viable for a short period, following encapsulation and intraperitoneal transplantation in rat. Other experimental conditions need to be tested to prevent or delay a decrease in hepatocyte specific gene expression.

To evaluate new therapies for human fulminant hepatic failure, a suitable large animal model is needed. The purpose of this study was to develop a reversible surgical model of acute hepatic liver failure by transient ischemia in pigs.Under general anesthesia, an end-to-side portacaval shunt was performed in 17 pigs and tape was laid around the hepatoduodenal ligament. Two days after construction of the functional portacaval shunt, 13 ambulant pigs underwent transient total liver ischemia by tightening of the tape around the hepatoduodenal ligament for 5.5 h. During ischemia, 10% glucose was continuously infused intravenously to prevent hypoglycemia.Ten animals (77%) died with hepatic coma after a mean duration of 22.5 +/- 1.9 h. The 3 remaining animals survived more than 5 days and were sacrificed. In dying animals, encephalopathy was observed 14 +/- 1.7 h after the onset of ischemia. During ischemia, similar progressive decrease of fibrinogen, platelets, prothrombin time, and factors V and VII activities was observed in dying and surviving animals. Just before death, mean prothrombin and factors V and VII activities were respectively 22 +/- 2, 21 +/- 4.4, and 24 +/- 5%. At 22 h, plasma ammonia and lactate levels were respectively 705 +/- 93 micromol/L and 10.5 +/- 0.4 mmol/L in dying animals and 249 +/- 75 micromol/L and 2.9 +/- 0.1 mmol/L in surviving animals (P0.01). Estimation of the percentage liver cells necrosed was 74 +/- 4.7% in the survivors and 86 +/- 5.5% in animals who died of hepatic coma (NS).This model is reproducible and reversible and should allow the quantitative evaluation of new technologies, such as bioartificial liver, for the support of hepatic failure in humans.

The general aim is to prepare a bioartificial liver to treat acute hepatic failure using allo- and xenogeneic hepatocytes, immunoprotected by macroencapsulation and transplanted into the peritoneal cavity. The goal of this study was to prepare a large amount of isolated porcine hepatocytes, to encapsulate them within biocompatible membranes for transplant in allo- and xenogeneic combinations and to examine the viability and functionality of the cells 6 weeks later. Hepatocyte isolation was performed in 12 kg pigs (n = 15) by dissociation of the liver with collagenase D (1 g) without oxygenation. Encapsulation of the hepatocyte suspension (10(7)/mL) was performed in hydrogel membranes AN69; hollow fibers (2 m x 0.8 mm) and flaskes (1.8 cm), and transplanted to Yucatan pigs (n = 4) and Lewis rats (n = 12). Six weeks later, they were removed to study the cell viability by histological examination, and the production of albumin by immunonephelometry. The rate of isolated hepatocytes was 38 +/- 5 x 10(9)/mL by liver of pig and the mean viability was 93 +/- 2%. Six weeks after transplantation, hepatocytes were viable, organized in lobules, and showed conserved albumin production. The same results were observed for allogenic and xenogeneic combinations. In conclusion, this method of liver dissociation allowed for preparation of a large amount of isolated hepatocytes from a single pig liver, theoretically sufficient to treat a patient with acute liver failure. Hydrogel membranes were well tolerated and allowed immunoprotection without immunosuppression. Transplanted hepatocytes remained functional. This work is an important step in progress toward clinical application.

Hepatocyte transplantation could be an alternative to whole liver transplantation. Allogeneic hepatocytes are rejected if transplanted without immunosuppression. The aim of this study was to transplant allogeneic hepatocytes in the peritoneum and to protect them from rejection by encapsulation in a new semi-permeable membrane.rats hepatocytes were encapsulated in hydrogel-based hollow fibers, obtained from AN69 polymer, before being transplanted into the peritoneum of rats. Outcome of allogeneic hepatocytes encapsuled in hollow fibers was compared to that of free allogeneic hepatocytes. Cell viability was assessed by erythrosine exclusion, morphology by electronic microscopy and function by albumin release.Up to 90 days, viability of allogeneic hepatocytes in hollow fibers was above 80%. The morphology remained normal at electronic microscopy. Albumin release was 16.5 +/- 0.3 (day 15), 14.2 +/- 2.0 (day 30), 8.8 +/- 0.1 (day 60) and 11.4 +/- 0.3 mg/24h/10(6) hepatocytes (day 90). Free hepatocytes did not survive at day 15.Viability and function of encapsulated allogeneic hepatocytes were maintained up to 90 days after transplantation, without immunosuppression.

Hepatocyte transplantation could be an alternative to whole liver transplantation. Allogeneic hepatocytes are rejected if transplanted without immunosuppression. The aim of this study was to transplant allogeneic hepatocytes in the peritoneum and to protect them from rejection by encapsulation in a new semi-permeable membrane.Rats hepatocytes were encapsulated in hydrogel-based hollow fibers, obtained from AN69 polymer, before being transplanted into the peritoneum of rats. Outcome of allogeneic hepatocytes encapsulated in hollow fibers was compared to that of free allogeneic hepatocytes. Cell viability was assessed by erythrosine exclusion, morphology by electronic microscopy and function by albumin release.Up to 90 days, viability of allogenic hepatocytes in hollow fibers was above 80%. The morphology remained normal at electronic microscopy. Albumin release was 16.5 +/- 0.3 (day 15), 14.2 +/- 2.0 (day 30), 8.8 +/- 0.1 (day 60) and 11.4 +/- 0.3 micrograms/24 h/10(6) hepatocytes (day 90). Free hepatocytes did not survive at day 15.Viability and function of encapsulated allogeneic hepatocytes were maintained up to 90 days after transplantation, without immunosuppression.

Hepatocytes normally express few major histocompatibility complex (MHC) class I and no MHC class II molecules, a phenomenon which could explain their low immunogenicity. However, in pathological situations, such as allograft rejection and cholestasis, hepatocytes strongly express MHC class I molecules and their immunogenicity could be different. The aim of this study was to assess the role of MHC expression on the immunogenicity of hepatocytes in vivo. Hepatocytes were obtained from normal and cholestatic DA rats by whole-liver perfusion with EDTA. Cholestasis was induced by ligation-section of the common bile duct. MHC expression on hepatocytes was assessed by cytofluorimetry after labelling with monoclonal antibodies against MHC class I and class II antigens. The percentage of hepatocytes expressing MHC class I was 9.8 ± 2.2% in normal rats and 77.2 ± 3.3% in cholestatic rats (P = 2 × 10 −4 ); MHC class II expression was present on 1 ± 0.5% of normal hepatocytes and 0.4% ± 0.1% of cholestatic hepatocytes (P > 0.05). Lewis rats received a DA or Wistar-Furth heart allograft 7 days after intravenous injection of 2 × 10 7 hepatocytes from normal or cholestatic DA rats. The DA heart allograft was rejected in 6.3 ± 0.4 days in Lewis controls, 8.8 ± 1.1 days (N.S.) in Lewis recipients that received normal DA hepatocytes and 17.6 ± 3.0 days (P = 2 × 10 −4 ) in Lewis recipients that received hepatocytes from cholestatic DA rats. The Wistar-Furth heart allografts were rejected in 9.8 ± 0.4 and 9.7 ± 0.4 days (P > 0.05), respectively, in Lewis controls and those with cholestatic DA hepatocytes. These results confirm that hepatocytes are normally poorly immunogenic in vivo and suggest that they become tolerogenic when MHC class I expression is induced. This could explain, at least partially, the relative tolerance of liver allografts.

The aim of this work was to evaluate the effect of intrasplenic hepatocellular transplantation on hepatic encephalopathy in an experimental model of chronic liver failure induced by end-to-side portacaval shunt in the rat. Inbred male Wistar Furth rats were divided into three groups: rats subjected to portacaval shunt (n = 10), rats subjected to portacaval shunt and intrasplenic hepatocellular transplantation of 10(7) hepatocytes isolated from livers of syngeneic rats (n = 10) and sham-operated rats (n = 10). Behavior tests were performed in a blind fashion at 3 wk, at 2 mo and at 3 mo after surgery. Spontaneous activity and nose-poke exploration by individual rats were studied in automated open field boxes equipped with infrared cells. Each cell beam interruption was automatically recorded on a microcomputer and transformed into a score index (counts/hour). Plasma levels of amino acids, ammonia and total biliary acids were measured. Portacaval shunt rats showed reduced spontaneous activity and nose-poke exploration scores. Intrasplenic hepatocellular transplantation significantly increased spontaneous activity after 2 mo and improved nose-poke exploration after 3 wk. At 3 mo, spontaneous activity and nose-poke exploration in portacaval shunt/intrasplenic hepatocellular transplantation rats were not significantly different from those of sham rats. Increases in plasma ammonia levels after portacaval shunt were not corrected. Amino acid imbalance and bile acid concentration in plasma were partially corrected by intrasplenic hepatocellular transplantation. These data show that intrasplenic hepatocellular transplantation can correct the neurological symptoms of hepatic encephalopathy in an experimental model of chronic liver failure and suggest that intrasplenic hepatocellular transplantation might be of therapeutic interest in chronic liver failure.

The liver is the most frequent site of metastases from colon cancer. To improve our knowledge of liver metastases and to develop new adjuvant therapies, a good animal model is necessary. The aims of this study were to obtain a model of liver metastases with intraportal injection of colon adrenocarcinoma cell aggregates (DHDK12 cell line) and to study the effect of various factors, i.e., sex, liver regeneration and immunosuppression, on the development of liver metastasis. Cell aggregates were injected into the portal vein of 59 syngenic male and female BD IX rats following randomization into three groups. Group 1, (control 12 males and 10 females) received only cell aggregates; group 2 (12 males and 10 females) underwent a 70% hepatectomy before cell injection; group 3 (15 males and 10 females) received cyclosporin A injections at a dose of 10 mg/kg per day for 28 days following cell injection. Autopsy was performed at 10 weeks. Liver metastases were more frequent in the male rats in group 3 than in those in group 1 (80% vs. 30%, p less than 0.04). The rate of liver metastases in females was not increased by immunosuppression (22.2% vs. 12.5%, N.S.). Liver resection (group 2) did not significantly modify the incidence of liver metastasis. No female had liver metastases in this group. This relatively simple model rapidly produces liver metastasis with a high yield, but only in male rats. Besides sexual factors, immunosuppression also increased the rate of experimental liver metastasis, while liver regeneration failed to do so.

The aim of this study was to assess directly the function of isolated hepatocytes 1 year after transplantation into the spleen, using an original model of isolated rat-spleen perfusion. Three specific liver functions, albumin synthesis, indocyanine-green clearance, and antipyrine oxidation, were studied. Five x 10(6) isolated hepatocytes were injected into the spleen of syngenic Wistar-Furth rats. One year later, splenectomy was performed, and the splenic pedicle was carefully isolated in order to allow a selective ex vivo perfusion for 3 hr. De novo albumin synthesis was studied by qualitatively using immunoelectrophoresis and autoradiography, and quantitatively using (35S)-methionine incorporation in albumin. De novo albumin synthesis was observed in spleens containing transplanted hepatocytes but not in controls (P less than 0.001); (35S)-methionine incorporation was significantly higher in spleens containing transplanted hepatocytes than in controls (132 +/- 67 cpm/spleen/hr vs. 14 +/- 6 cpm/spleen/hr, P less than 0.001). Antipyrine clearance was significantly higher in spleens with transplanted hepatocytes than in controls (67.4 +/- 4.9 microliters/min/g vs. 0.2 +/- 0.4 microliters/min/g, P less than 0.01). No statistically significant difference was observed with indocyanine-green clearance (4.2 +/- 6.0 microliters/min/g, vs. 5.2 +/- 5.1 microliters/min/g, P greater than 0.05); this was probably due to the absence of compartmentation between the sinusoid and biliary sectors in this model. In conclusion, using this original isolated rat-spleen perfusion model, it was directly observed that 1 year after transplantation, intrasplenic hepatocytes can perform two liver-specific functions, i.e., de novo albumin synthesis and antipyrine clearance.

The aim of this work was to assess the effect of intrasplenic liver cell transplantation (ILCT) on hepatic insufficiency induced by a terminolateral portocaval shunt (PCS) in rats. Thirty syngenic Wistar Furth rats were divided up into three groups: (a) rats with PCS (n = 10); (b) rats with PCS then ILCT of 10(7) liver cells isolated from the livers of syngenic rats (n = 10); (c) operated control rats (n = 10). Double-blind behavior tests were carried out two weeks, two months and six months after surgery. The spontaneous motor activity and the exploring activity of each rat were studied in automated cages fitted with infrared diodes. Each interruption of the infrared beam was automatically recorded by a computer and converted into an activity score (number/hour). The spontaneous motor activity and the exploring activity were poor in the rats with PCS. The ILCT significantly increased the spontaneous motor activity and the exploring activity 2 months and 3 weeks after transplantation, respectively. Three months after transplantation, the spontaneous motor activity and the exploring activity in the PCS/ILCT group were not significantly different from those of the control rats. This study shows that ILCT can correct the neurological signs of hepatic encephalopathy in an experimental model of chronic hepatic insufficiency, and suggests that ILCT may produce therapeutic benefits in chronic hepatic insufficiency.