Liver cell membrane alloantigens as cellular markers in genotypic mosaic rat livers undergoing chemically induced hepatocarcinogenesis (original) (raw)
Related papers
Transplant International, 1999
Hepatocyte transplantation is a conceptually attractive alternative to whole organ grafting for some inborn metabolic errors and for fulminant liver failure. However, studies of the immunogenicity of transplanted allogeneic hepatocytes have yielded contradictory results. In these experiments, the effect of purification and cryopreservation of the hepatocytes on the ability of these cells to engraft in the mouse allogeneic recipients without immunosuppression was studied. BALB/ cByJ mouse crude (unpurified), modified (purified or cryopreserved), or dead (irradiated) hepatocyte preparations labeled with fluorescein dye CFSE were infused either into the portal vein or into the spleen parenchyma of the recipient CBA mice. A histological examination revealed normal appearance of engrafted modified hepatocytes with no signs of acute rejection up to 21 days posttransplant. Many of the intrasplenically implanted hepatocytes migrated into the hepatic sinusoids. The modified hepatocytes showed intact ultrastructural appearance 7 days after transplantation. The numbers of inoculated crude hepatocytes rapidly declined with signs of dense infiltration of mononuclear cells in the graft indicating destructive response. The fluorescence of dead hepatocytes was undetectable. These results suggest that reduced immunogenicity may be responsible for the longer survival time of inoculated, purified or cryopreserved hepatocytes with no adverse morphological effects.
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.
Revista Española de Enfermedades Digestivas, 2019
Background: the shortage of donors of hepatocyte transplantation therapy led to the use of so-called marginal donors. Some donors may have a hepatic illnesses that is associated with hepatic preneoplasia with foci of altered hepatocytes (FAH). Aims: to determine whether recipients developed FAH upon transplantation with hepatocytes from a preneoplastic liver and whether FAH progresses to a preneoplastic hepatocyte-derived tumor (PHDT), up to 60 days after transplantation. Material and methods: male Wistar adult rats were used as donors and recipients. Donors underwent a 2-phase model of liver preneoplasia for hepatocyte isolation. Recipients underwent a partial two thirds hepatectomy and received 150,000 hepatocytes. Recipients were euthanized seven and 60 days after transplantation. The number of FAH per liver area, percentage of liver occupied by FAH, the hepatic enzymatic profile, the percentage of prothrombin time (PT), the proliferative index (PI) and liver morphology were analyzed. Results: recipients developed few and very isolated FAH. No statistical differences were found between hepatic enzyme activities and PT. There were no differences between the groups with regard to the number of FAH per liver area and percentage of liver occupied by FAH after 60 days. The PI decreased on day 60 compared to day seven. No morphological alterations were found. Conclusions: recipients developed few FAH that did not increase in number or size, nor did they progress to PHDT and had normal plasma biochemical features and liver morphology up to 60 days post-transplant. Additional studies are needed to determine whether FAH development constitutes a risk for recipients while waiting for whole organ transplant.
Isolation and culturing of hepatocytes from human livers
Journal of Tissue Culture Methods, 1992
Using a modified collagenase digestion procedure, we were successful in the isolation of viable hepatocytes from human liver surgical wastes, unused transplantable livers, and diseased livers from transplant recipients. The modified procedures for isolation involved collagenase perfusion of a sample of limited size (<50 g) with only one cut surface, perfusion via only one blood vessel, the use of an appropriate amount of collagenase, as well as manual "massaging" of the liver sample during critical stages of the collagenase perfusion. Using this modified procedures, we had close to 100% success rate in the isolation of viable hepatocytes from the human liver samples.
Maintenance of adult rat hepatocytes on C3H/10T1/2 cells
Cancer research, 1979
A procedure is described for maintaining primary cultures of adult rat hepatocytes on a layer of irradiated C3H/10T1/2 cells. These hepatocytes were capable of metabolizing the liver carcinogen N-2-acetylaminofluorene to water-soluble products and after 14 days in culture could still metabolize approximately 70% of the Day 1 level. Hepatocytes maintained on the C3H/10T1/2 cells were inducible for the liver-specific enzyme tyrosine aminotransferase, and exhibited approximately a 4-fold induction by hydrocortisone during a 10-day culture period. Morphologically, these hepatocytes retained many characteristics of hepatocytes in vivo. By contrast, hepatocytes maintained on plastic lost both N-2-acetylaminofluorene-metabolizing ability and tyrosine aminotransferase activity by Day 5. This was presumably due to degeneration of the hepatocytes and an overgrowth by fibroblasts. The maintenance of morphologically and biochemically functional hepatocytes in culture on feeder cells may provide...
Cell Transplantation, 1994
Large numbers of human hepatocytes were obtained from split and whole livers by using an adaptive form of the collagenase perfusion technique employed in rodent and human biopsies. In order to guarantee a homogenous distribution of the perfusate within the whole specimen, major hepatic veins were cannulated with large bore catheters. This technique allowed for the isolation of human hepatocytes on a large scale (up to 18.5 × 109 in one case) from normal and diseased liver specimens. The yield of isolated normal viable hepatocytes is inversely proportional to the donor age. In addition, it was noted that a short time between declared death and organ harvest (cross clamp time) results in higher viability of hepatocytes. In contrast, the time of cold organ preservation did not correlate with the viability or the yield of isolated hepatocytes. We conclude that the technique presented here allows isolation of large numbers of human hepatocytes from specimens unsuitable for transplantatio...
Morphological and biochemical characterization of a human liver in a uPA-SCID mouse chimera
Hepatology, 2005
A small animal model harboring a functional human liver cell xenograft would be a useful tool to study human liver cell biology, drug metabolism, and infections with hepatotropic viruses. Here we describe the repopulation, organization, and function of human hepatocytes in a mouse recipient and the infections with hepatitis B virus (HBV) and hepatitis C virus (HCV) of the transplanted cells. Homozygous urokinase plasminogen activator (uPA)-SCID mice underwent transplantation with primary human hepatocytes, and at different times animals were bled and sacrificed to analyze plasma and liver tissue, respectively. The plasma of mice that were successfully transplanted contained albumin and an additional 21 human proteins. Liver histology showed progressive and massive replacement of diseased mouse tissue by human hepatocytes. These cells were accumulating glycogen but appeared otherwise normal and showed no signs of damage or death. They formed functional bile canaliculi that connected to mouse canaliculi. Besides mature hepatocytes, human hepatic progenitor cells that were differentiating into mature hepatocytes could be identified within liver parenchyma. Infection of chimeric mice with HBV or HCV resulted in an active infection that did not alter the liver function and architecture. Electron microscopy showed the presence of viral and subviral structures in HBV infected hepatocytes. In conclusion, human hepatocytes repopulate the uPA+/+-SCID mouse liver in a very organized fashion with preservation of normal cell function. The presence of human hepatic progenitor cells in these chimeric animals necessitates a critical review of the observations and conclusions made in experiments with isolated “mature” hepatocytes. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index.html). (HEPATOLOGY 2005;41:847–856.)
The fate of hepatocyte cell line derived from a liver injury model with long-term in vitro passage
Molecular & Cellular Toxicology, 2018
Backgrounds: orthotopic liver Transplantation (olT) is the therapy of choice for the treatment of end-stage liver disease, but the severe shortage of organ donors, complex and expensive surgical procedure and increased mortality of prospective organ recipient limit the use of olT. To overcome this problem the technique of hepatocyte transplantation has been considered as an alternative to olT. Hepatocyte transplantation is less invasive, cost effective cryo-preservable and can be distributed from single donor to multiple recipients. in this study we have established and characterize the hepatocyte cell line possessing the morphological and functional characteristics of hepatocytes from chemically injured liver. Hence we hypothesized that the hepatocyte cell line derived from liver injury can be used for hepatocyte transplantation. Methods: To induce the priming of hepatocyte, mice were fed with 3,5-diethoxycarbonyl-1,4-dihydro-collidine (DDC) diet for 3 weeks and hepatocytes were obtained by two step collagenase perfusion method, hepatocytes hence obtained was expended by >300 passages and tested for various hepatocyte specific functions. Results: The cell line derived from liver injury model retains morphological and functional characteristics of hepatocytes in long-term in vitro culture. These cells transplanted to mice showed significant survival rate. Conclusion: The Hepatocyte cell line derived from liver injury model were used for hepatocyte transplantation and showed the significantly higher survival rate.
PubMed, 1985
An improved transplantation system for the study of carcinogen-altered hepatocytes is described. This system, which is based on that reported by Laishes and Farber (Cancer Res., 61: 507-512, 1978), involves the transfer of hepatocytes from male F344 animals to syngeneic adult hosts. Unlike the earlier protocol, the recipient rats were fed phenobarbital (PB) rather than the DNA-reactive agent, 2-acetylaminofluorene. gamma-Glutamyl transpeptidase (GGT)-positive hepatocytes were induced in the donor animals by one of three different hepatocarcinogenic treatment regimens. The recipient rats received 0.05% PB in the diet for 3 wk prior to the cell transfer and were maintained on the PB diet for 2 to 7 mo. Hepatocytes from a male F344 donor rat that had received a 70% hepatectomy and 30 mg of diethylnitrosamine per kg and had been maintained on 0.05% PB for 12 mo formed GGT-positive colonies and hepatocellular carcinomas in both male and female recipients. No GGT-positive colonies were formed when 0.05% PB was omitted from the diet of the recipients. A 70% partial hepatectomy of the recipients at the time of cell transfer was also essential for the development of colonies and tumors. The mean volume of the colonies was 5 times larger in female recipients than in males, occupying 38% of the total liver volume in the females. GGT-positive foci arose in recipient livers that had received hepatocytes from either a male F344 donor rat treated according to the Solt and Farber [Nature (Lond.), 263: 701-703, 1976] selection protocol or a male F344 donor rat that received a 70% hepatectomy and 30 mg of diethylnitrosamine per kg and was maintained on 0.05% PB for 5 mo. The recipient animals were treated with PB which the initial experiments showed was essential for the development of foci. The number and volume of the foci in the recipient varied according to the treatment regimen that the donor rat received. This system provides a method for analyzing the growth regulation of altered foci at various stages of neoplastic development during hepatocarcinogenesis in the rat in the absence of DNA-reactive selection agents.
Toxicology in Vitro, 1995
The aim of this study was to compare human hepatocytes isolated from livers accepted and from livers discarded for transplantation with respect to viability and drug transport function. In addition, the influence of age of the donor and preservation time of the liver on cell viability was determined. Cell viability was assessed by trypan blue exclusion, MTT reduction, morphological integrity and ATP content, and drug transport function by uptake and excretion of taurocholic acid. Hepatocytes could be isolated successfully from livers accepted as well as from livers discarded for transplantation, with a median yield of 5.0 x 106 cells/g (range 0.1 to 42.4) and 0.7 x IO6 cells/g (range 0.0 to 22.7), respectively (not significantly different). These cells were not significantly different with respect to viability and transport rate of taurocholate. Neither the age of the donor nor the duration of liver preservation (643 hr in University of Wisconsin solution) significantly influenced cell yield and viability. It is concluded that because of this overlap in cell viability, hepatocytes isolated both from accepted and from discarded livers can in principle be used to investigate drug transport functions in the human liver.