Eliane Alexandre

Evaluation of the effect of culture configuration on morphology, survival time, antioxidant status and metabolic capacities of cultured rat hepatocytes.

We evaluated the antioxidant status, namely cellular lipid peroxidation, by measuring thiobarbituric acid reactive substances (TBARS), cellular reduced glutathione (GSH) content, glutathione reductase (GSSG-R), glutathione transferase (GST), glutathione peroxidase (GSH-Px) and catalase activities in rat liver, hepatocytes immediately after isolation and in two-dimensional (2D) culture (on non-coated or collagen-coated dishes, as collagen-collagen or collagen-Matrigel sandwich cultures) or three-dimensional (3D) culture on Matrigel-coated dishes. Microsomal cytochrome P450 (CYP)- and UDP-glucuronosyl transferase (UGT)- dependent activities were also assessed in rat livers and hepatocyte cultures. The overall antioxidant status of rat hepatocytes immediately after isolation was not significantly different from that of rat livers. During culture, GSH was increased in 2D but not in 3D cultures in accordance with morphological observations; that is that matrix-cell interactions involving GSH, important in 2D, are minimal in 3D cultures. While UGT- and GST-dependent activities were equivalent in cultured hepatocytes and in rat livers, both catalase and GSH-Px activities decreased with time in all culture configurations. Constitutive CYP-dependent activities were drastically decreased in hepatocytes after isolation and attachment and did not recover in any culture configuration tested. Our results highlight that, although 2D sandwich cultures and 3D cultures on Matrigel allow longevity of rat hepatocyte cultures and optimal induction of CYPs, an imbalance in phase I/phase II detoxication processes in cultured rat hepatocytes occurs, whatever the culture configuration.

Protective effects of N-acetylcysteine on hypothermic ischemia-reperfusion injury of rat liver.

We investigated whether intraportal injection of 150 mg/kg N‐acetylcysteine (NAC) into rats reduced hepatic ischemia‐reperfusion injury after 48 hours of cold storage and 2 hours of reperfusion. The organ was isolated and perfused to evaluate liver function. The control group received an intraportal injection of 5% dextrose. NAC increased L‐cysteine concentrations 15 minutes after injection (1.29 ± 0.11 μmol/g vs. 2.68 ± 0.4 μmol/g,P < .05). However, neither treatment modified glutathione liver concentrations relative to preinjection values. After 48 hours of cold storage and 2 hours of reperfusion, livers from NAC‐treated rats produced larger amounts of bile than those in the control group (5.04 ± 1.92 vs. 0.72 ± 0.37 μL/g liver; P < .05), and showed a significant reduction in liver injury, as indicated by reduced release of lactate dehydrogenase (679.4 ± 174.4 vs. 1891.3 ± 268.3 IU/L/g; P < .01), aspartate transaminase (AST) (13.94 ± 3.5 vs. 38.75 IU/L/g; P < .01), alanine transaminase (ALT) (14.92 ± 4.09 vs. 45.91 ± 10.58 IU/L/g; P < .05), and acid phosphatase, a marker of Kupffer cell injury (344.4 ± 89.6 vs. 927.3 ± 150.8 IU/L/g; P < .01) in the perfusate. Reduced glutathione concentrations in the perfusate were similar in the two groups (805 ± 69 vs. 798 ± 252 nmol/L/g), whereas oxidized glutathione (GSSG) concentrations were higher in the control group (967 ± 137 vs. 525 ± 126 nmol/L/g; P < .05). Reduced glutathione (GSH) concentrations in liver tissue collected at the end of perfusion were significantly higher in the NAC group (7.3 ± 0.9 vs. 4.1 ± 1.0 μmol/g; P < .05). The protective effect of NAC on cold ischemia‐reperfusion liver injury persisted when animals were pretreated with buthionine sulfoximine (BSO), a specific inhibitor of glutathione synthesis. Our results suggest that NAC enhances the concentrations of cysteine within hepatocytes, providing a substrate for glutathione synthesis during reperfusion. They also indicate that NAC has a direct protective effect on Kupffer cells, which are the first source of reactive oxygen intermediates during reperfusion. (HEPATOLOGY 1995; 22:539–545.)

Tissue collection, transport and isolation procedures required to optimize human hepatocyte isolation from waste liver surgical resections. A multilaboratory study

Abstract: Background: The European Center for Validation of Alternative Methods (ECVAM) has funded a prevalidation study in three laboratories (France, USA and UK) on the use of human hepatocyte cultures to predict cytochrome P‐450 induction.

Hepatocyte ploidy in normal young rat

The aim of the present study was to examine the relation between hepatocyte size and ploidy in Sprague-Dawley rat liver. Therefore, subpopulations of hepatocytes of various sizes were separated from the isolated crude hepatocyte population either mechanically or by using centrifugal elutriation. Hepatocyte size was determined on scanning electron microscopy photographs. Ploidy of hepatocytes was assessed by flow cytometry. The crude hepatocyte population was very heterogeneous in sizes, with diameters ranging from 8 to 39 microm. Hepatocyte ultrastructure was well preserved as demonstrated by transmission electron microscopy. The distribution of hepatocytes within the ploidy classes was the following: 19.6+/-3.6% diploid, 56.2+/-3.2% tetraploid and 3.4+/-0.6% octoploid mononucleated cells. Thus approximately 79% of hepatocytes appeared mononucleated. The binucleated hepatocytes (21%) had two diploid nuclei (18.7+/-2.9%) or two tetraploid nuclei (2.1+/-0.6%). A similar distribution of hepatocytes into ploidy classes was obtained in subpopulations of hepatocytes of various sizes. Our findings suggest that distribution into ploidy classes is not strictly correlated with hepatocyte size. In accordance with previous observations, our results on hepatocyte ploidy from periportal or perivenous origin using digitonin perfusion, is in favour of the existence of ploidy zonation within the rat hepatic lobule.

Efficacy of preconditioning with N-acetylcysteine against reperfusion injury after prolonged cold ischaemia in rats liver in which glutathione had been reduced by buthionine sulphoximine

OBJECTIVE To investigate the ability of N-acetylcysteine (NAC) to prevent cold ischaemic-reperfusion injury and improve hepatic integrity in a glutathione-depleted condition. DESIGN Open laboratory study. SETTING University hospitals, Japan and France. MATERIALS 40 male Wistar rats. INTERVENTIONS To produce a glutathione-depleted liver, buthionine sulphoximine (BSO) was injected intraperitoneally 2 hours before either NAC or 5% dextrose was infused 15 minutes before the liver was harvested. We used an isolated perfused rat liver model that had undergone prolonged hypothermic ischaemia, cold-storage for 48 hours and reperfusion for 120 minutes. MAIN OUTCOME MEASURES Concentrations of hepatic enzymes released into samples of perfusate, concentration of adenosine triphosphate in liver tissue, concentrations of reduced and oxidized glutathione in perfusate, and bile production. RESULTS The concentrations of the hepatocellular enzymes and oxidised glutathione in the perfusate samples were significantly reduced in the NAC group compared with the 5% dextrose group. Bile production improved significantly in the NAC group compared with the 5% dextrose group. The concentration of reduced glutathione in liver tissue was not increased by NAC. CONCLUSION In a glutathione-depleted liver NAC prevented hepatic injury and improved liver integrity after a cold ischaemic-reperfusion injury, by acting not as a substrate for glutathione synthesis but as a direct free radical scavenger.

Engraftment and albumin production of intrasplenically transplanted rat hepatocytes (Sprague-Dawley), freshly isolated versus cryopreserved, into Nagase analbuminemic rats (NAR).

Banking of cryopreserved hepatocytes is a prerequisite for large-scale hepatocyte transplantation in the clinic. We compared the efficacy of intrasplenic transplantation into Nagase analbuminemic rats (NAR) of freshly isolated (FIH) and cryopreserved (CH) hepatocytes. Hepatocytes were cryopreserved using a controlled rate freezing protocol. Albumin production of thawed CH and FIH was measured in vitro in culture by ELISA and by Western blot. After in vivo intrasplenic transplantation of NAR with either FIH or CH we assessed 1) albumin in the serum of recipients by ELISA and by Western blotting analysis at different time intervals, and 2) hepatocyte engraftment by albumin immunohistochemical staining into spleens and livers at euthanasia. In vitro, albumin was produced up to day 4 of culture in both CH and FIH. In vivo, no intrasplenic engraftment of hepatocytes occurred. Intrahepatic engraftment of CH (cell number/mm2) was significantly (twofold) lower than that of FIH and appeared only as isolated cells and small (<10 cells) clusters, while bigger clusters (>10 cells) were observed with FIH. In the FIH group, serum albumin production was observed up to 32 – 49 days posttransplantation while in the CH group no serum albumin production was detected. Our results emphasize the need to improve 1) hepatocyte transplantation procedures either by repeated hepatocytes injections and/or by transplantation under a regeneration response, and 2) the freeze/thaw protocols of hepatocytes.

Comparative evaluation of celsior solution versus viaspan in a pig liver transplantation model

BACKGROUND In a pig liver transplantation model, we compared the effects of Celsior solution (CS), an extracellular preservation solution, with Viaspan (University of Wisconsin solution, UW) on graft function and animal survival. METHODS Pig livers were flushed with either CS or UW solution and cold-stored for 12 hr (group 1) or for 8 to 10 hr (group 2). Grafts were transplanted orthotopically. Intrahepatic reduced and oxidized glutathione and adenine nucleotides were evaluated 1 hr after reperfusion. Liver function of transplanted animals was monitored for up to 6 days by serum transaminases, total bilirubin, purine nucleoside phosphorylase, and prothrombin levels. RESULTS In group 1, all animals died within 24 hr after reperfusion regardless of the preservation solution used. In group 2, no significant difference was seen in survival between the CS (72%) and the UW (67%) groups 6 days after transplantation, and there were no statistically significant differences in the biochemical data. There were no differences in histological evaluation of the livers at the time of death or killing of the animals between the CS and UW groups. CONCLUSION Within the limits of this pilot study, CS is equivalent to UW in terms of graft function and animal survival.

Influence of Pre-, Intra- and Post-Operative Parameters of Donor Liver on the Outcome of Isolated Human Hepatocytes

The aim of the present study was to analyse, retrospectively on a large panel of patients (149), the influence of the donor liver characteristics on the outcome of human hepatocyte isolation obtained from resected liver biopsies from surgical waste after hepatectomy. Among the pre-operative parameters, the type of disease, age and sex of the patient, previous chemotherapy, alcohol or tobacco consumption did not affect the yield, viability, attachment rate and function of the isolated human hepatocytes. Pre-operative biological and anatomopathological data indicated that, while mild steatosis (≤10% steatotic hepatocytes) did also not affect the outcome of hepatocyte isolation, stronger steatosis (>10% steatotic hepatocytes) tended to decrease hepatocyte yield. Cholestasis, as assessed by γ-glutamyl transferase serum values, significantly negatively correlated with the percentage of digested liver and the yield of viable cells. Intra-operative clamping time, that is, warm ischaemia, longer than 30 min was found to decrease both the percentage of digested liver and cell yield. Among the post-operative parameters, the percentage of digested liver decreased when biopsy weights were higher than 100 g, the use of glue tended to increase both the percentage of digested tissue and the yield of viable cells.In conclusion, human diseased livers appear to be a valuable source of isolated functional human hepatocytes. We recommend, for an optimal isolation, to use liver biopsies weighing less than 100 g, to glue the section surfaces of the biopsies and to avoid the use of moderate steatotic livers (>10% steatotic hepatocytes) and cholestatic livers, as well as livers undergoing warm ischaemia or clamping during resection due to the decrease in cell yield.

Metabolic capacities in cultured human hepatocytes obtained by a new isolating procedure from non-wedge small liver biopsies:

A new isolating procedure of human hepatocytes has been developed using two-step collagenase digestion by a nonperfusion procedure (NP) of non-wedge liver biopsies. 1. A yield of 2 - 76106 hepatocytes/g liver, 52 - 95% viability and 13 - 75% attachment were obtained from liver biopsies weighing 6 - 60 g, comparable to that obtained when using the classical perfusion procedure (P) to isolate human hepatocytes from wedge liver samples of 50 - 150 g. 2. In culture, human hepatocytes obtained by NP remained attached to plastic for up to 5 days and displayed the usual morphological characteristics. Their metabolic capacities, assessed by liver-specific albumin and urea synthesis and by CYP-dependent and conjugation pathways, were equivalent to those of human hepatocytes obtained by P. In addition, they responded adequately to specific CYP inducers, demonstrating that they constitute a model in which human drug metabolism and toxicity studies can be performed.

Expression of cytochromes P-450 2E1, 3A4 and 1A1/1A2 in growing and confluent human HepG2 hepatoma cells effect of ethanol

In cultured human hepatoma HepG2 cells, cytochrome (CYP) 1A-associated 7-ethoxyresorufin-O-deethylase (EROD), CYP 3A-associated benzyloxyresorufin O-debenzylase (BROD) and CYP 2E1-associated p-nitrophenol-hydroxylase (PNPH) decreased during time in culture. The enzyme activities in cells at confluence were 35-60% of the activities in cells 24 hours after seeding. Similarly, CYP 3A and CYP 2E1 proteins were present at higher concentrations in growing (G) than in confluent (C) HepG2 cells. CYP 1A1/1A2 protein was not detected, neither in G nor in C HepG2 cells but was strongly induced by 3-methylcholanthrene (3-MC) treatment. Ethanol (EtOH) was shown to increase CYP 2E1 and CYP 3A proteins and CYP 1A1/1A2-, CYP 2E1- and CYP 3A-associated mixed-function oxidase activities (MFOs) in HepG2 cells, as has been previously reported for primary cultures of human hepatocytes. These effects were observed only at the beginning of culture, in growing HepG2 cells, demonstrating the influence of the growth stage of HepG2 cells on their response to EtOH treatment. This is, to our knowledge, the first report on increases in CYP proteins and associated MFOs by EtOH in HepG2 cells. It suggests that growing HepG2 cells provide a useful in vitro model system in which to study the regulation of human CYPs by EtOH.