Repeated intraportal hepatocyte transplantation in analbuminemic rats (original) (raw)
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A Simple and Effective Method to Improve Intrasplenic Rat Hepatocyte Transplantation
Cell Transplantation, 2004
Transplanted hepatocytes integrate, survive, and express their specific functions in the liver parenchyma. The aim of this study was to determine whether a large number of hepatocytes could move from the spleen to the liver when the cells are injected together with sodium nitroprusside, and if the improved hepatocyte migration may be related with portal vein dilatation. Wistar rats were transplanted in the spleen with fluorescent-labeled hepatocytes alone or together with sodium nitroprusside. At 1, 3, 6, and 24 h after the transplant, the liver from recipient animals was removed and morphometric analyses were performed. Portal and arterial pressures were also measured immediately after intrasplenic injection of a solution of sodium nitroprusside, hepatocytes alone, or hepatocytes plus sodium nitroprusside. Intrasplenically injected sodium nitroprusside produced a transient drop in arterial pressure and a sustained reduction in portal pressure. During hepatocyte transplantation it i...
Transplantation of isolated hepatocytes
Journal of Hepatology, 1988
Over the past decade there has been an increase in interest in studies of transplantation of isolated hepatocytes. The survival of hepatocytes in sites distant from the normal hepatic circulation has provided insights into cell proliferation and phenotypic expression of liver-specific functions. The possibility of treating liver insufficiency in disease has also been investigated. The present review is intended to give an account of the development of methods for the study of hepatocyte transplantation, techniques for assessment of graft function and future possibilities for application of the technique. (A) Methods used to isolate viable hepatocytes Continuous portal perfusion Most experiments on hepatocyte transplantation
Intrasplenic transplantation of allogeneic hepatocytes prolongs survival in anhepatic rats
Hepatology, 1998
To examine whether hepatocytes transplanted in the spleen can function as an ectopic liver, we performed hepatocyte transplantation in rats that were rendered anhepatic. Total hepatectomy was performed by using a novel single-stage technique. Following hepatectomy, Group 1 rats (n ؍ 16) were monitored until death to determine survival time without prior intervention. Group 2 anhepatic rats (n ؍ 20) were sacrificed at various times to measure blood hepatocyte growth factor (HGF) and transforming growth factor 1 (TGF-1) levels. Group 3 (n ؍ 16) rats received intrasplenic injection of isolated hepatocytes (2.5 ؋ 10 7 cells/rat) followed by total hepatectomy after 3 days. Group 4 (n ؍ 12) sham-transplanted rats received intrasplenic saline infusion, and after 3 days they were rendered anhepatic. Group 2, 3, and 4 rats were maintained on daily Cyclosporine A (10 mg/kg; intramuscularly). Group 1 anhepatic rats survived for 22.4 ؎ 5.2 hours (standard deviation). The anhepatic state was associated with a progressive and statistically significant rise in blood HGF and TGF-1 levels. Rats that received hepatocyte transplantation before total hepatectomy had a significantly longer survival time than sham-transplanted anhepatic controls (34.1 ؎ 8.5 vs. 15.5 ؎ 4.8 hrs, P F .01). Additionally, at 12 hours post-hepatectomy, transplanted rats had significantly lower blood ammonia, prothrombin time, international normalized ratio, and TGF-1 levels when compared with sham-transplanted controls. In conclusion, intrasplenic transplantation of allogeneic hepatocytes prolonged survival, improved blood chemistry, and lowered blood TGF-1 levels in rats rendered anhepatic. (HEPATOLOGY 1998;28: 1365-1370.) Transplantation of isolated hepatocytes (HcTx) has been shown to ameliorate, at least partially, specific enzymatic defects 1-4 and provide temporary support to animals with experimentally induced liver failure. 5-10 Preliminary results with HcTx in humans are also encouraging. 11-13 These studies indicate that transplanted hepatocytes can assume intact whole liver functions. However, whereas in the case of a genetic liver defect, the appearance of a missing gene product can be attributed solely to HcTx, the mechanism by which transplanted hepatocytes support recipients with physical liver injury (viral, toxic, ischemic) remains obscure. The observed beneficial effects, e.g., improved blood chemistry and neurological status, can be ascribed to transplanted cell function, to improved function of the native liver, or both. We have recently studied this problem and shown, for the first time, that in rats with fulminant hepatic failure (FHF), ectopically (spleen) implanted syngeneic hepatocytes improved regeneration of the native liver. 14 In the present study, we describe a novel single-stage technique of total hepatectomy (TH) in the rat and show that in rats rendered anhepatic, intrasplenic HcTx prolonged survival, improved blood chemistry, and partially corrected imbalance between blood levels of hepatocyte growth factor (HGF) and transforming growth factor 1 (TGF-1). MATERIALS AND METHODS Animal studies were conducted according to the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health. Animals Adult male Sprague Dawley rats weighing 150 to 350 gm (Harlan Sprague-Dawley, Inc., Indianapolis, IN) were used. Rats were acclimatized to our laboratory conditions for 1 week before use in the experiments. They were housed in a climate-controlled (21°C) room under a 12-hour light/dark cycle. Rats were given tap water and commercial rat chow (Rodent Chow 5001, Ralston Purina, St. Louis, MO) ad libitum. All operations were performed under methoxyflurane (Mallinckordt Veterinary Inc., Mundelein, IL) general anesthesia with use of sterile surgical technique. All animals were warmed externally (heating lamp) during surgery and then until recovery from anesthesia. Chemicals Unless otherwise noted, all chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). Hepatocyte Isolation Donor hepatocytes were isolated from the livers of Sprague-Dawley rats (ϳ150g) by in situ two-step ethylenediaminetetraacetic acid/collagenase digestion, as described previously. 15 Hepatocytes were further purified by sedimentation on Percoll density gradient (Pharmacia Biotech., Piscataway, NJ). Final hepatocyte viability was always greater than 90%, as determined by trypan blue exclusion.
Intraportal hepatocyte transplantation in the pig: a hemodynamic and histopathological study1
Transplantation, 2002
Background. Hepatocyte transplantation is an attractive treatment for various liver diseases. The intraportal route of transplantation is favored, but little information is available on the possible adverse effects in this technique. We investigated the influence of intraportal loads of hepatocytes on portal, pulmonary, and systemic hemodynamics in 13 pigs. Methods. Under general anesthesia, pigs were provided with an arterial line, a Swan-Ganz catheter, and two intraportal catheters, one for cell infusion and one for heparin infusion and portal pressure measurement. Pig hepatocytes were infused at a rate of 25 million cells/min. Results. The first six animals were used to develop the infusion technique. In the last seven animals, portal pressure increased linearly with cell load upon infusion of 400-2400؋10 6 hepatocytes (r 2 ;407.0؍ P<0.05). Portal flow measured by Doppler sonography decreased by 23-66% below basal values. An inverse linear relationship was found between portal pressure and portal flow (r 2 ;976.0؍ P<0.05), portal flow approaching zero for portal pressure >40 mmHg. Pulmonary arterial pressure increased by 11-62%. AST increased up to 10-fold, and platelets decreased by 22-58%. Hepatocytes-containing thrombi were present in segmental and in smaller portal branches. Hepatocytes were always identified in lung sinusoids 48 hr after infusion, and a small basal pulmonary infarction was found in one animal. Conclusions. These data suggest that up to 2.4% of total hepatocyte mass can be infused in this large animal model. However, the risk of significant thrombotic complications should be considered for clinical applications. 1 Supported by grants from the Regione del Veneto and the Fondazione per l'Incremento dei Trapianti d'Organo, Padova, Italy.
Cell Transplantation, 2011
The efficiency of hepatocyte transplantation into the liver varies with the method of administration. This study investigated whether retrograde infusion via the hepatic vein provides a sufficient number of donor cells for the liver. Donor hepatocytes were isolated from dipeptidyl peptidase IV (DPPIV +) rats and transplanted into DPPIV − rat livers either by antegrade portal vein infusion or retrograde hepatic vein infusion. Hepatocyte engraftment ratios and localization were evaluated by histological DPPIV enzymatic staining at 1 week and 8 weeks after the transplantation. No significant differences in engraftment efficiency were observed at either 1 week or 8 weeks after transplantation by either route. However, the localization of the transplanted hepatocytes differed with the administration route. Portal vein infusion resulted in predominantly periportal engraftment, whereas hepatic vein infusion led to pericentral zone engraftment. Immunohistochemical analysis showed that the transplanted hepatocytes engrafted in the pericentral zone after retrograde infusion displayed intense CYP2E1 staining similar to the surrounding native hepatocytes. CYP2E1 staining was further enhanced by administration of isosafrole, an inducing agent for various cytochrome P450 enzymes, including CYP2E1. This study demonstrates a novel approach of transplanting hepatocytes into the liver through retrograde hepatic vein infusion as the means to target cell implantation to the pericentral zone.
Cell Transplantation, 2011
Although hepatocyte transplantation (HCTx) is expected to become a useful therapy for human liver diseases, allogenic hepatocytes still tend to be rejected within a short period due to host immunosurveillance. In the present study, we investigated the effect of prior bone marrow transplantation (BMTx) for the engraftment of allogenic hepatocytes using the analbuminemic rat transplantation model. The hepatocytes of Lewis (LEW) rats were not accepted in the liver of retrorsine (RS)/partial hepatectomy (PH)-treated analbuminemic F344 (F344-alb) rats, which express the disparate major histocompatibility complex (MHC) against that of LEW rats. Prior BMTx with the LEW bone marrow cells (BMCs) after sublethal irradiation achieved acceptance and repopulation of LEW hepatocytes in the liver of the RS/PH-treated F344-alb rats, associated with elevation of serum albumin. The replacement of hepatic parenchyma with albumin positive (Alb +) donor hepatocytes and elevation of serum albumin levels were dependent on the bone marrow reconstitution by donor BMCs, which was more efficiently achieved by intrabone marrow (IBM)-BMTx than by intravenous (IV)-BMTx. Our results demonstrate that efficient bone marrow reconstitution by IBM-BMTx enables the replacement of the hepatic parenchyma with allogenic hepatocytes in RS/PH-treated analbuminemic rats without immunosuppressants.
Amplification of Engrafted Hepatocytes by Preparative Manipulation of the Host Liver
Artificial Organs, 2001
Scarcity of donor livers is a major obstacle to the general application of hepatocytes for the development of bioartificial liver assist devices as well as intracorporeal engraftment of hepatocytes for the treatment of inherited metabolic diseases. The number of hepatocytes that can be transplanted into the liver safely in a single sitting also limits the utility of this procedure. These limitations could be addressed by providing preferential proliferative advantage to the transplanted cells. Studies using transgenic mouse recipients or donors have indicated that massive repopulation of the host liver by engrafted hepatocytes requires that the transplanted cells are subjected to a proliferative stimulus to which the host hepatocytes cannot respond. Prevention of host hepatocyte proliferation has been achieved by treatment with a plant alkaloid, retrorsine. Because retrorsine is carcinogenic, we have evaluated
Hepatology, 1999
These rats served as hepatocyte recipients. Syngeneic F344 rats that served as hepatocyte donors were obtained commercially (Taconic Farms, Germantown, NY). The animals were housed under 14/10 hour light/dark cycles, respectively. The protocols were approved by the Animal Care and Use Committee at the Albert Einstein College of Medicine. The animal use was in accordance with the National Research Council' s Guide for the Care and Use of Laboratory Animals (NIH Publication, 1985). The studies used multiple animals in groups of at least 3 to 6 rats each. After hepatocyte transplantation, recipient animals were killed at 2, 4, 6, 8, 12, 16, 20, and 24 hours, and at 2, 3, 4, and 7 days for tissue analysis. These animals received 2 ϫ 10 7 hepatocytes via the spleen. To show the duration of portal hypertension after cell transplantation, one animal group was subjected to portal pressure measurements before, during, and immediately after transplantation of 2 ϫ 10 7 cells, with repeat measurements 3 hours after cell transplantation. These animals served as their own controls. Portal pressures in other animals were studied only once after 1, 3, 7, 10, and 21 days of cell transplantation immediately before portal venography. Although these animals constituted two groups, one receiving 2 ϫ 10 7 cells and the other, 7.5 ϫ 10 7 cells via the spleen, data are presented only from the former group, because the findings were generally similar. In addition, normal animals that did not receive cells were included as controls for comparing portal pressures and vascular anatomy with hepatocyte recipients. Multiple additional rats were examined at 2 and 24 hours after cell transplantation for electron microscopic studies. Cell Isolation and Culture. Hepatocytes were isolated by in situ collagenase perfusion of the liver as previously described. 4,7 In brief, the liver was perfused for the first 5 minutes with buffer containing 0.5 mmol/L EGTA (ethylene glycol-bis(-aminoethyl ether)-N,N,NЈNЈ-tetraacetic acid), pH 7.4 at a rate of 20 mL/min, followed by perfusion for 1 minute with buffer lacking EGTA, and finally with buffer containing 0.025% (wt/vol) collagenase D in the presence of 2