Cryopreservation Of Hepatocytes Research Articles

Multiple intrasplenic hepatocyte transplantations in the dalmatian dog

Background: Hepatocyte transplantation is an attractive potential treatment for liver-based inborn errors of metabolism and for fulminant hepatic failure. Dalmatian dogs have a metabolic error that results in hyperuricosuria. This report focuses on the effect of multiple, sequential intrasplenic transplants of fresh and cryopreserved hepatocytes in dalmatians. Methods: Dalmatians underwent intrasplenic hepatocyte transplantation with hepatocytes taken from healthy mongrels. Dalmatian urinary uric acid excretion was measured preoperatively, and this served as the control value. Three hepatocyte transplantations were performed at 30-day intervals—the first with freshly isolated cells,and both the second and the third with cryopreserved hepatocytes from the same donor. Urinary uric acid excretion was measured postoperatively twice per week. Results: The urinary uric acid excretion decreased an average of 54% after the first hepatocyte transplantation. The effect was transient and lasted an average of 22 days (range, 19-50 days). Subsequent intrasplenic hepatocyte transplantation with cryopreserved hepatocytes resulted in similar decreases in urinary uric acid excretion. Each transplant resulted in a significant decrease in urinary uric acid excretion when compared with baseline values (P = <.001). Conclusions: Sequential intrasplenic hepatocyte transplantation is feasible in this model. This method provided a significant, but transient, correction in urinary uric acid excretion that was similar with either fresh or cryopreserved hepatocytes. A substantial biologic effect provided by cryopreserved hepatocytes has important implications in clinical hepatocyte transplantation. (Surgery 2000;127:193-9.)

CRYOPRESERVED PRIMARY HEPATOCYTES AS A CONSTANTLY AVAILABLE IN VITRO MODEL FOR THE EVALUATION OF HUMAN AND ANIMAL DRUG METABOLISM AND ENZYME INDUCTION*

The use of primary hepatocytes is now well established for both studies of drug metabolism and enzyme induction. Cryopreservation of primary hepatocytes decreases the need for fresh liver tissue. This is especially important for research with human hepatocytes because availability of human liver tissue is limited. In this review, we summarize our research on optimization and validation of cryopreservation techniques.The critical elements for successful cryopreservation of hepalocytes are (1) the freezing protocol, (2) the concentration of the cryoprotectant [10% dimethylsulfoxide (DMSO)], (3) slow addition and removal of DMSO, (4) carbogen equilibration during isolation of hepatocytes and before cryopreservation, and (5) removal of unvital hepatocytes by Percoll centrifugation after thawing. Hepatocytes of human, monkey, dog, rat, and mouse isolated and cryopreserved by our standard procedure have a viability ≥ 80%. Metabolic capacity of cryopreserved hepatocytes determined by testosterone hydroxylation, 7-ethoxyresorufin-O-de-ethylase (EROD), 7-ethoxycoumarin-O-deethylase (ECOD), glutathione S-transferase, UDP-glucuronosyl transferase, sulfotransferase, and epoxide hydrolase activities is ≥60% of freshly isolated cells. Cryopreserved hepatocytes in suspension were successfully applied in short-term metabolism studies and as a metabolizing system in mutagenicity investigations. For instance, the complex pattern of benzo[a]pyrene metabolites including phase II metabolites formed by freshly isolated and cryopreserved hepatocytes was almost identical. For the study of enzyme induction, a longer time period and therefore cryopreserved hepatocyte cultures are required. We present a technique with cryopreserved hepatocytes that allows the induction of testosterone metabolism with similar induction factors as for fresh cultures. However, enzyme activities of induced hepatocytes and solvent controls were smaller in the cryopreserved cells.In conclusion, cryopreserved hepatocytes held in suspension can be recommended for short-term metabolism or toxicity studies. Systems with cryopreserved hepatocyte cultures that could be applied for studies of enzyme induction are already in a state allowing practical application, but may be further optimized.

Investigation of functional and morphological integrity of freshly isolated and cryopreserved human hepatocytes.

There is a pressing need for alternative therapeutic methods effective in the treatment of patients with liver insufficiency. Isolated human hepatocytes may be a viable alternative or adjunct to orthotopic liver transplantation in such patients. The purpose of this study was to evaluate the viability and functional integrity of freshly isolated and cryopreserved human hepatocytes, in preparation for a multi-center human hepatocyte transplantation trial. We are currently processing transplant-grade human parenchymal liver cells from nondiseased human livers that are obtained through a network of organ procurement organizations (OPOs). Thus far, sixteen hepatocyte transplants have been performed using hepatocytes processed by our methods. At the time of referral all specimens were deemed unsuitable for transplantation due to anatomical anomalies, high fat content, medical history, etc. Hepatocytes were isolated from encapsulated liver sections by a modified two-step perfusion technique. Isolated cells were cryopreserved and stored in liquid nitrogen for one to twelve months. The total yield of freshly isolated hepatocytes averaged 3.7x10(7) cells per gram of wet tissue. Based on trypan blue exclusion, fresh preparations contained an average of 85% viable hepatocytes vs. 70% in cryopreserved samples. The plating efficiencies of cells seeded immediately after isolation ranged from 87% to 98%, while those of cryopreserved/thawed cells were markedly lower. Flow cytometry analysis of cells labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) showed that there was no significant difference in viability compared with trypan blue staining. Both freshly isolated hepatocytes and those recovered from cryopreservation showed typical and intact morphology as demonstrated by light and electron microscopy. The product of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reaction was always expressed more intensely in cultures of freshly isolated hepatocytes. Measurements of lactate dehydrogenase (LDH) leakage were inversely correlated with trypan blue exclusion and CFSE labeling. Energy status, evaluated by the intracellular ATP concentration measurements, and various liver-specific functions such as urea synthesis and metabolism of 7-ethoxycoumarin were maintained both in fresh and cryopreserved/thawed hepatocytes. However, the activities were expressed at different levels in thawed cells. These data illustrate the importance and feasibility of human hepatocyte banking. In addition, it is clear that further refinements in the methods of hepatocyte isolation and cryopreservation are needed to utilize more fully these valuable cells in the clinic.

PREDICTION OF IN VIVO HUMAN HEPATIC CLEARANCE IN DRUG DISCOVERY

We have investigated a quantitative prediction of in vivo human hepatic clearance from in vitro experiments. In vivo intrinsic clearance (CLint, in vivo) were compared with in vitro intrinsic clearance (CLint, in vitro) from in vitro metabolism data obtained by using liver microsomes focusing on P450 metabolism in rats, dogs and humans. The scaling factor (CLint, in vivo/CLint, in vitro) were different among the model compounds, while most of the animal scaling factors were within 2-fold of the values in humans. When human CLint, in vitro values were compared with the actual CLint, in vivo, correlations were not necessarily good. By contrast, using human CLint, in vitro corrected with the animal scaling factor yielded better predictions of CLint, in vivo that were mostly within 2-fold of actual values. Further, CLint, in vivo were compared with CLint, in vitro from in vitro metabolismd ata obtained by using rat fresh and human cryopreserved hepatocytes. Human CLint, in vitro corrected with the rat scaling factor yielded good predictions of CLint, in vivo that were mostly less than several-fold of actual values. This method thus provides a more reliable prediction of human hepatic clearance and will be useful in drug discovery.

Induction of cytochrome-P450 in cryopreserved rat and human hepatocytes

Our laboratory has been routinely using suspended and cultured human hepatocytes for predicting drug metabolism and enzyme induction by drug candidates to aid drug discovery. Increasing limitation and irregular availability of human tissue has indicated the need for maximizing the use of this valuable resource. Cryopreservation of surplus hepatocytes after isolation would greatly increase the potential of this model. However, cryopreservation of hepatocytes by various methods has resulted in cells with poor metabolic activity and unacceptably low survival rates in culture. Recently Zaleski et al. (Biochem. Pharmacol. 46 (1993) 111–116) reported that cryopreserved rat hepatocytes retained metabolic capacity similar to fresh hepatocytes when the cells were preincubated for 30 min at 37°C in Krebs–Ringer bicarbonate buffer prior to freezing. To further explore this methodology, both the functional capacity of the cells in culture as well as their ability to retain CYP inducibility was investigated with thawed cryopreserved hepatocytes. Although human hepatocytes were used in this study the initial work focused on rat hepatocytes as a cell model. Our results showed that while the preincubation step did not appear to effect the initial viability of cryopreserved hepatocytes, survival of the cells in culture was greatly enhanced. Plating efficiencies for nonpreincubated cryopreserved hepatocytes were decreased to ≈15% of fresh cells after 48 h in culture. In contrast, cells that had been preincubated prior to freezing had an excellent plating efficiency (∼60%) and responded to classical CYP inducers dexamethasone, β-naphthoflavone and phenobarbital in a manner indistinguishable from that of fresh hepatocytes. Experiments with human hepatocytes has also demonstrated similar results. This is the first time to our knowledge that cryopreserved hepatocytes from both rat and human have been shown to reproducibly respond to CYP inducers in culture.

Cryopreserved human hepatocytes: characterization of drug-metabolizing activities and applications in higher throughput screening assays for hepatotoxicity, metabolic stability, and drug–drug interaction potential

Cryopreserved human hepatocytes were extensively characterized in our laboratory. The post-thaw viability, as determined by Trypan blue exclusion, ranged from, the viability, measured via dye exclusion, ranged from 55 to 83% for hepatocytes cryopreserved from 17 donors. Post-thaw viability and yield (viable cells per vial) were found to be stable up to the longest storage duration evaluated of 120 days. Drug-metabolizing enzyme activities of the cryopreserved hepatocytes (mean of ten donors) as percentages of the freshly isolated cells were: 97% for cytochrome P450 isoform (CYP) 1A2, 78% for CYP2A6, 96% for CYP2C9, 86% for CYP2Cl9, 90% for CYP2D6, 164% for CYP3A4, 76% for UDP-glucuronidase, and 88% for umbelliferone sulfotransferase. Known species-differences in 7-ethoxycoumarin (7-EC) metabolism were reproduced by cryopreserved hepatocytes from human, rat, rabbit, dog, and monkey, illustrating the utility of cryopreserved hepatocytes from multiple animal species in the evaluation of species-differences in drug metabolism. Higher throughput screening (HTS) assays were developed using cryopreserved human hepatocytes for hepatotoxicity, metabolic stability, and inhibitory drug–drug interactions. Dose-dependent cytotoxicity, measured using MTT metabolism as an endpoint, was observed for the known hepatotoxic chemicals tamoxifen, clozapine, cadmium chloride, diclofenac, amiodarone, tranylcypromine, precocene II, but not for 2-thiouracil. Cell density- and time-dependent metabolism of 7-EC and dextromethorphan were observed in the HTS assay for metabolic stability. Known CYP isoform-specific inhibitors were evaluated in the HTS assay for inhibitory drug–drug interactions. Furafylline, sulfaphenazole, quinidine, and ketoconazole were found to be specific inhibitors of CYP1A2, CYP2C9, CYP2D6, and CYP3A4, respectively. Tranylcypromine and diethyldithiocarbamate were found to be less specific, with inhibitory effects towards several CYP isoforms, including CYP2A6, CYP2C9, CYP2Cl9, and CYP2E1. These results suggest that cryopreserved human hepatocytes represent a useful experimental tool for the evaluation of drug metabolism, toxicity, and inhibitory drug–drug interaction potential.

Comparative effects of rifabutin and rifampicin on cytochromes P450 and UDP-glucuronosyl-transferases expression in fresh and cryopreserved human hepatocytes

The aim of this study was to evaluate rifabutin (RBT) and rifampicin (RIF) capabilities in inducing various xenobiotic metabolizing enzymes such as cytochromes P450 (CYPs) and UDP-glucuronosyl-transferases (UGTs) in cultured fresh and cryopreserved human hepatocytes. Enzyme induction was assessed through the use of several diagnostic markers, i.e. testosterone, midazolam (MDZ), diazepam (DZP) and 7-ethoxyresorufin for CYP-dependent enzyme reactions; and AZT for UGT-dependent enzyme reactions. RBT concentrations (0.118, 0.708 μM) were selected according to previously published pharmacokinetic data in patients. The known CYP3A4 inducer in humans, RIF, was used as a positive control. At the concentrations used, no sign of cytotoxicity was evidenced. Both compounds were able to dose-dependently induce the overall metabolism of testosterone (∼2-fold for RBT, ∼4-fold for RIF) and the formation of the 6β-hydroxylated-derivative (up to ∼4-fold over control for RBT and ∼10-fold for RIF), which is CYP3A4 dependent. The other hydroxylated metabolites (16α-OH and 2α-OH) were also enhanced. The metabolism of MDZ, which is specifically metabolized by CYP3A4 in humans, was also investigated following drug's exposure to hepatocytes. DZP one, which is governed by various CYPs, including CYP3A, was also investigated. RBT was shown to increase the biotransformation of both benzodiazepines (∼1.9-fold over control). Moreover, the effects of both drugs on ethoxyresorufin O-deethylase activity (EROD), which is representative of CYP1A1/2 isoforms, were tested. Results showed only a moderate induction of this marker (∼2-fold over control) when compared to the high effect observed after hepatocyte exposure to 3-methylcholantene (∼14-fold over control). Finally, the action of RBT and RIF on UGTs expression was investigated by using AZT as diagnostic substrate: glucuronides formation was not significantly affected by the two rifamycin derivatives. On the whole, exposure of fresh or cryopreserved human hepatocytes to RBT dose-dependently affected the levels of drug metabolizing enzymes in a dose-dependent manner. However, as already demonstrated by in vivo pharmacokinetic studies, its inducing properties towards CYPs, CYP3A in particular, are less pronounced than RIF.

Overview: hepatocytes and cryopreservation—a personal historical perspective

Hepatocytes represent an important tool for the investigation of species differences in drug metabolism and toxicity. Data obtained with hepatocytes from multiple animal species, including man, allow better prediction of the effects of xenobiotics in man. Cryopreservation of hepatocytes extends the use of this important experimental system by enhancing the convenience of its use. Also, it allows the researchers to perform experiments not plausible with freshly isolated hepatocytes, such as the direct comparison of xenobiotic toxicity and metabolism in hepatocytes from multiple human donors in a single experiment.

Present status of the application of cryopreserved hepatocytes in the evaluation of xenobiotics: consensus of an international expert panel

Successful cryopreservation of freshly isolated hepatocytes would significantly decrease the need for freshly-procured livers for the preparation of hepatocytes for experimentation. Hepatocytes can be prepared, cryopreserved, and used for experimentation as needed at different times after isolation. Cryopreservation is especially important for research with human hepatocytes because of the limited availability of fresh human livers. Based on the cumulative experience of this international expert panel, a consensus was reached on the various aspects of hepatocyte cryopreservation, including cryopreservation and thawing procedures and applications of the cryopreserved hepatocytes. Key to successful cryopreservation includes slow addition of cryopreservants, controlled-rate freezing with adjustment for the heat of crystallization, storage at −150°C, and rapid thawing. There is a general consensus that cryopreserved hepatocytes are useful for short-term xenobiotic metabolism and cytotoxicity evaluation.

Hepatocyte transplantation for the treatment of human disease.

A great deal of work with animal models indicates that hepatocytes transplanted into the liver or spleen survive, function, and participate in the normal regenerative process. Recent clinical studies suggest that hepatocyte transplantation may be useful for bridging patients to whole organ transplantation and for providing metabolic support during liver failure and for replacing whole organ transplantation in certain metabolic liver diseases. In specific situations where the rate of death of host hepatocytes is high, the transplanted cells can repopulate the native liver. Techniques have been established for the large scale isolation, culture and cryopreservation of human hepatocytes. Shortage of donor organs and the need for immunosuppression are two major hurdles to widespread application of this procedure, and current research in experimental animals is aimed at addressing these problems.

Effects of Cell Concentration on Viability and Metabolic Activity during Cryopreservation

Increasing the cell concentration during the cryopreservation of red blood cells (RBC) increased hemolysis. Similarly, increasing the cell concentration during the cryopreservation of hepatocytes reduced both the viability of the cells as assessed by trypan blue exclusion and the metabolic activity of the trypan blue-excluding cells. In both cell types, significant damage appeared at cell concentration levels exceeding 60%. With tightly packed RBC, hemolysis reached 63%, while for hepatocytes after thawing and dilution, trypan blue viability was reduced to 41% of that of the cells initially isolated. The viability of cryopreserved hepatocytes, estimated by trypan blue exclusion, was considerably reduced following incubation for 1 h at 37°C and the extent of this reduction was also a function of cell concentration during freezing and thawing. The trypan blue viability of hepatocytes that were tightly packed during cryopreservation and then incubated at a concentration of 5 × 106viable cells/ml fell dramatically to only 12.5%. The protein-synthesizing activity and the membrane transport activity of these cells, expressed in terms of cells that excluded trypan blue immediately following thawing and removal of the cryoprotectant, were reduced to 38.7 and 33.5%, respectively, of the activity in cells that were packed at 10% cell concentration. The fall in metabolic activity may have been due to complete loss of activity in some of the cells or reduced activity in most or all of the cells or any combination of these factors. It is concluded that there may be many mechanisms involved, but the most important factor is probably stresses produced by unphysiological cell–cell contacts occurring during the freeze–thaw cycle.

Metabolic activity of fresh and cryopreserved dog hepatocyte suspensions

1. Dog hepatocytes were cryopreserved at 6 × 106 viable cells/ml in a suspension buffer containing 10% DMSO and were stored in liquid nitrogen. 2. The exclusion of trypan blue dye was 96± 2 and 85± 9% in fresh and cryopreserved (CP) hepatocytes, respectively. Albumin synthesis was unaffected by freezing. 3. Ethoxycoumarin and ethoxyresorufin O-deethylase activities were equivalent in fresh and CP hepatocytes. 4. The profile of testosterone metabolism was unaffected by freezing. Total hydroxylase activities were 815± 33 pmol/min/106 CP cells in freshly isolated whole hepatocytes and 463± 24 pmol/min/106 CP whole hepatocytes, but they were equivalent in fresh and CP hepatocyte homogenates supplemented with 250 μM NADPH. 5. Phase 2 enzymes were functional in freshly thawed CP hepatocytes but they required exogenous addition of cofactors (20 μM UDPGA and 1.7 μM PAPS). 6. When placed in suspension for longer times, fresh and CP cell viabilities were 88± 6 and 64± 2% after 4 h. ECOD and EROD activities were equivalent in fresh and CP hepatocyte suspensions, over 4 h. Testosterone hydroxylase activities were well maintained in fresh cell suspensions but they declined to 63± 6% of the initial activity after 4 h in CP hepatocytes. 7. These results indicate that CP dog hepatocytes are a suitable in vitro system for xenobiotic metabolisms in ce enzyme functions in CP hepatocytes were stabilized. Cofactors in freshly thawed CP hepatocytes should be measured and controlled for optimal use.

Influence of Alginate Gel Entrapment and Cryopreservation on Survival and Xenobiotic Metabolism Capacity of Rat Hepatocytes

Rat hepatocytes immobilized in calcium–alginate beads were tested for their ability to survive and functionin vitroby comparison with hepatocyte monolayer cultures before and after cryopreservation. The freeze–thaw protocol previously designed for suspended hepatocytes was used; the freezing medium was the Leibovitz medium containing 10% fetal calf serum and 16% dimethylsulfoxide. The functions investigated included protein, lipid, and urea synthesis, albumin secretion, glycogen content, and various phase I and phase II enzyme activities. Cell viability was not altered by immobilization and the freeze–thaw procedure. Most functions tested were expressed at similar levels in unfrozen immobilized hepatocytes and corresponding hepatocyte monolayers. Only protein neosynthesis and some phase II drug metabolizing enzyme activities were decreased. The freeze–thaw process had no detrimental effect, whatever the function investigated. In addition, cryopreserved immobilized hepatocytes remained responsive to inducers: ethoxyresorufinO-deethylase activity was increased 11–12 times by 3-methylcholanthrene treatment in both fresh and cryopreserved alginate-entrapped hepatocytes. These results clearly show that hepatocytes immobilized in calcium–alginate beads remain functional even after cryopreservation indicating that they represent a promising approach for xenobiotic metabolism and toxicity studies.

Large scale cryopreservation of porcine hepatocytes for the clinical treatmentof acute liver failure

Large scale cryopreservation of porcine hepatocytes for the clinical treatmentof acute liver failure

Cryopreservation of rat hepatocyte monolayer cultures.

1. Most previous attempts to cryopreserve hepatocytes have used suspensions stored at either -70 degrees C or in liquid nitrogen, and the major problem is that these do not, on subsequent thawing, attach well in culture. This limits their use in studies of drug metabolism and xenobiotic-induced toxicity. In this manuscript we demonstrate successful cryopreservation of rat hepatocytes as monolayers attached to a collagen film. 2. Monolayers can be frozen and thawed without significant loss of cells, and although damage to the internal and plasma membranes is evident immediately post-thaw, a remarkable repair process takes place over 24-48 h post-thaw. Immediately post-thaw only 10% of the cells exclude Trypan Blue, but by 48 h 80-90% of the thawed cells are viable, indicating that repair of the plasma membranes has taken place. 3. The cells post-thaw retain aspects of liver-specific function including cytochrome P450 content and albumin synthesis. However, cytosolic proteins are lost through the damaged membranes and, probably because of this, urea synthesis from ammonia is retained at only 25% of pre-freeze values. 4. A cryopreservation method based on adherent hepatocytes on a collagen substrate overcomes the problems encountered with culture of cryopreserved hepatocyte suspensions, and may provide a practical means of establishing a 'bank' of hepatocytes from several donors and species.

2. 肝細胞の凍結保存(平成7年度第41回低温生物工学会研究報告)

2. 肝細胞の凍結保存(平成7年度第41回低温生物工学会研究報告)

Effective cryopreservation and long-term storage of primary human hepatocytes with recovery of viability, differentiation, and replicative potential

Despite reports of successful cryopreservation of primary human hepatocytes, existing methods do not produce sufficient recovery of viable cells to meet the needs of basic research or clinical trials of hepatocellular transplantation. We now describe a protocol for efficient cryopreservation of primary human hepatocytes using University of Wisconsin (UW) solution, fetal bovine serum, and dimethyl sulfoxide (DMSO). This method provides >90% viability of differentiated, primary human hepatocytes 8 mo after cryopreservation as measured by trypan blue exclusion, preserves hepatocyte morphology, liver-specific gene expression (α 1 antitrypsin), and replication. The effectiveness of UW solution as a cryopreservative agent suggests that metabolic as well as ultrastructural factors may be important in the effective cryopreservation of primary human hepatocytes. The present method represents an effective protocol for cryopreserving differentiated primary human hepatocytes for research. This method may allow characterization and banking of human hepatocytes for clinical applications, including hepatocellular transplantation and hepatic assist devices.

Effective cryopreservation and long-term storage of primary human hepatocytes with recovery of viability, differentiation, and replicative potential

Despite reports of successful cryopreservation of primary human hepatocytes, existing methods do not produce sufficient recovery of viable cells to meet the needs of basic research or clinical trials of hepatocellular transplantation. We now describe a protocol for efficient cryopreservation of primary human hepatocytes using University of Wisconsin (UW) solution, fetal bovine serum, and dimethyl sulfoxide (DMSO). This method provides > 90% viability of differentiated, primary human hepatocytes 8 mo after cryopreservation as measured by trypan blue exclusion, preserves hepatocyte morphology, liver-specific gene expression (alpha 1 antitrypsin), and replication. The effectiveness of UW solution as a cryopreservative agent suggests that metabolic as well as ultrastructural factors may be important in the effective cryopreservation of primary human hepatocytes. The present method represents an effective protocol for cryopreserving differentiated primary human hepatocytes for research. This method may allow characterization and banking of human hepatocytes for clinical applications, including hepatocellular transplantation and hepatic assist devices.

Effective cryopreservation and long-term storage of primary human hepatocytes with recovery of viability, differentiation, and replicative potential.

Despite reports of successful cryopreservation of primary human hepatocytes, existing methods do not produce sufficient recovery of viable cells to meet the needs of basic research or clinical trials of hepatocellular transplantation. We now describe a protocol for efficient cryopreservation of primary human hepatocytes using University of Wisconsin (UW) solution, fetal bovine serum, and dimethyl sulfoxide (DMSO). This method provides > 90% viability of differentiated, primary human hepatocytes 8 mo after cryopreservation as measured by trypan blue exclusion, preserves hepatocyte morphology, liver-specific gene expression (alpha 1 antitrypsin), and replication. The effectiveness of UW solution as a cryopreservative agent suggests that metabolic as well as ultrastructural factors may be important in the effective cryopreservation of primary human hepatocytes. The present method represents an effective protocol for cryopreserving differentiated primary human hepatocytes for research. This method may allow characterization and banking of human hepatocytes for clinical applications, including hepatocellular transplantation and hepatic assist devices.

Beneficial Effect of S-Adenosylmethionine during both Cold Storage and Cryopreservation of Isolated Hepatocytes

Oxygen free radicals appear to be the prime cell-toxic products during cold preservation. Glutathione (GSH) seems to play a critical role in cell protection against oxidant stress. The experimental decrease of intracellular GSH in vivo may be prevented by the administration of S -adenosylmethionine (SAMc), which seems also to play an important role in preserving the structure of cell membranes. We designed our study to investigate whether the addition of SAMe to EuroCollins solution (EC) could provide a similar degree of protection as the more complex University of Wisconsin (UW) solution during cold preservation. In adddion, we have investigated a possible protective action of SAMe during hepatocyte cryopreservation. Wistar rat hepatocytes ( 106 cells/ml) were stored in either EC (±12 μmol/l SAMe) or UW. In parallel, hepatocytes ( 106 cells/ml) were cryopreserved in M199 culture medium (±SAMe) using dimethyl sulfoxide as cryoprotectant. LDH release, viability and hepatocyte GSH and malondialdehyde (MDA) content were sequentially determined during cold preservation. There were no differences on viability or GSH and MDA content between EC + SAMe and UW stored cells, although LDH release was slightly higher in the first group. The addition of SAMe also attenuated the decrease in both viability (37 ± 0.8 vs 53.0 ± 7.4%. mean ± SEM, N = 5, P < (0.05) and GSH content (134 ± 15.1 vs 45.1 ± 16.8%. mean ± SEM. N = 5, P < 0.01), observed after thawing. Our results suggest that SAMe could be a useful additive for both cold storage and cryopreservation solutions of hepatocytes.