Monooxygenation, conjugation and other functions in cryopreserved rat liver slices until 24 h after thawing

For the extensive use of precision-cut liver slices (particularly of human origin) for toxicological investigations successful cryopreservation is necessary. But so far, survival of thawed slices was limited to few hours. This was now overcome by modification of previous procedures. The concentration of DMSO as a cryoprotectant was enhanced to 30%, and washing steps after rapid thawing were omitted. The slices were frozen in liquid nitrogen, thawed at 38 degrees C and incubated immediately in Williams medium E. Protein and potassium contents were stable until 24 h. Glutathione content, amounting to nearly 50% of fresh slices, increased during incubation. High initial lactate dehydrogenase leakage dropped after medium change to less than half during 2-24 h. Testosterone hydroxylation and 7-ethoxycoumarin O-deethylation rates were similar to fresh slices, the latter reaction was inducible by beta-naphthoflavone within 24 h. Methylumbelliferone glucuronidation and p-nitrophenol glucuronidation and sulfation were well measurable and either maintained or decreased by about 50% until 24 h.Altogether, the results are encouraging for further experiments to standardise cryopreservation conditions and to investigate the suitability of this cryopreservation protocol with human liver slices.     

COLD AND CRYOPRESERVATION OF MONKEY LIVER SLICES

Both cynomolgus and rhesus monkeys are utilized in chemical toxicity screening and drug development studies by industry and government laboratories. Along with better utilization of their tissue for in vivo studies, it would be advantageous if the tissue could be cold-preserved or cryopreserved for future in vitro experimentation. Therefore, livers were excised from control monkeys and precision-cut tissue slices were prepared. The objective of this study was twofold: to compare cold-preservation solutions (V-7 and Viaspan) and to compare controlled-rate and vitrification cryopreservation protocols. Monkey liver slices were cold-stored in V-7 or Viaspan preservation solutions for 7 days. V-7 maintained slice viability for 5 days whereas Viaspan maintained slice viability for 1 day. In the controlled-rate freezing procedure, slices were exposed to 10% dimethyl sulfoxide and 90% fetal calf serum (FCS); cooled at the rate of 0.5°C, 1°C, or 12°C per min to -70°C; then placed into liquid nitrogen. Vitrification was accomplished by exposing slices stepwise to increasing concentrations of 1,2-propanediol (1.2, 2.4, and 4 M) in FCS with direct submersion into liquid nitrogen. In both protocols, slices were rewarmed quickly to 37°C and then incubated in FCS for 4 h. Three viability parameters were used to measure slice viability - retention of potassium, leakage of lactate dehydrogenase, and synthesis of protein. Liver slices cryopreserved at a rate of 0.5°C per min and vitrified successfully retained 80 to 90% of their viability. These results confirm the feasibility of functional cold preservation and cryopreservation protocols for monkey liver slices that would allow for a more efficient use of monkey tissue.     

Cryopreservation of precision-cut rat liver slices using a computer-controlled freezer

Precision-cut liver slices are frequently used to study hepatic toxicity and metabolism of xenobiotics in vitro. Successful cryopreservation techniques will enhance an efficient and economic use of scarcely available (human) liver tissue. For primary hepatocytes, slow freezing has been accepted as the best approach towards successful cryopreservation. For slices, however, no agreement exists on the optimal way of cryopreservation and both slow and fast freezing techniques have been reported. The aim of the present study was to determine the applicability of a computer-controlled slow freezing technique for the cryopreservation of (rat) liver slices. Thus far, this technique has not been described in detail. Our studies confirmed that slow freezing was most successful in the cryopreservation of primary rat hepatocytes. Based on this observation, the slow freezing technique was applied to the cryopreservation of rat liver slices. Directly after thawing, slice viability was between 60 and 100% of fresh values, depending on the parameter determined. However, after additional culturing, slice viability was reduced. This decrease in slice viability was more pronounced in comparison to primary hepatocytes. In conclusion, the slow freezing technique was confirmed to be a successful approach for the cryopreservation of primary rat hepatocytes, and was found to be of limited use for the cryopreservation of rat liver slices.     

Phase I and phase II metabolic activities are retained in liver slices from mouse, rat, dog, monkey and human after cryopreservation.

Precision-cut liver slices are described as a valuable tool for in vitro metabolism studies of potential drug candidates. Recently, some papers reported successful cryopreservation conditions for liver slices, facilitating a broader and more efficient use of the tissue (particularly of human origin). The aim of this study is to evaluate the effect of cryopreservation on both phase I and phase II metabolism in liver slices prepared from mouse, rat, dog, monkey and human, using rapid freezing in the presence of 18% DMSO. Glucuronidation and sulfation activities (phase II) in both freshly prepared and cryopreserved liver slices were determined by rapid LC-MS/MS analyses using 7-hydroxycoumarin as a marker substrate. Testosterone was used as a marker substrate for cytochrome P450 mediated drug metabolism (phase I). Although the metabolic patterns and rates varied among the different species, the phase I and phase II metabolic capacities of the liver slices were well maintained after cryopreservation. Despite the good biotransformation capacity of cryopreserved slices a decrease in viability, expressed as ATP content and LDH leakage, was observed. MTT reduction was well maintained after cryopreservation. The possibility to cryopreserve liver slices will allow a more efficient utilisation of tissue, in particular from human, but also from dog and monkey. Finally, cryopreserved liver slices from mouse, rat, dog, monkey and human with good phase I and II metabolism activities are a useful in vitro tool to compare metabolite profiles of new chemical entities between species.     

High conservation of both phase I and II drug-metabolizing activities in cryopreserved rat liver slices

1. Xenobiotic-metabolizing enzymes, including both cytochrome P450 and phase II-conjugating systems, have been characterized in rat liver slices cryopreserved in 12 or 18% dimethylsulphoxide (DMSO). 2. Several cytochrome P450 isoforms in rat liver slices metabolized testosterone to a variety of hydroxylated products. The rates of formation of these same products were well maintained during cryopreservation of the slices in both 12 or 18% DMSO. 3. After cryopreservation of rat liver slices in 18% DMSO, the rates of metabolism of ropivacaine to 3-hydroxyropivacaine, 4-hydroxyropivacaine and PPX (all catalysed by different cytochrome P450 isoforms) were approximately 94, 79 and 82% respectively of the corresponding rates observed with fresh slices. 4. The rates of conjugation of 7-hydroxycoumarin and 1-naphthol by rat liver slices were significantly decreased after cryopreservation in 12% DMSO, but they were maintained when the concentration of this cryopreservant was increased to 18% 5. After cryopreservation in 12% DMSO, the mitochondrial reduction of the tetrazolium salt MTT by rat liver slices was significantly lowered. In contrast, slices cryopreserved in 18% DMSO demonstrated no significant decrease in their capacity to reduce MTT. 6. Thus, in agreement with previous studies, it was found that cytochrome P450-dependent activities are retained after cryopreservation of liver slices. Although phase II-conjugating enzyme activities are more sensitive to cryopreservation, it was shown that increasing the concentration of DMSO present during cryopreservation could circumvent the problem. This modification improves the usefulness of cryopreserved rat liver slices as a tool in drug metabolism studies.     

High conservation of both phase I and II drug-metabolizing activities in cryopreserved rat liver slices

1. Xenobiotic-metabolizing enzymes, including both cytochrome P450 and phase IIconjugating systems, have been characterized in rat liver slices cryopreserved in 12 or 18% dimethylsulphoxide (DMSO). 2. Several cytochrome P450 isoforms in rat liver slices metabolized testosterone to a variety of hydroxylated products. The rates of formation of these same products were well maintained during cryopreservation of the slices in both 12 or 18% DMSO. 3. After cryopreservation of rat liver slices in 18% DMSO, the rates of metabolism of ropivacaine to 3-hydroxyropivacaine, 4-hydroxyropivacaine and PPX (all catalysed by different cytochrome P450 isoforms) were C 94, 79 and 82% respectively of the corresponding rates observed with fresh slices. 4. The rates of conjugation of 7-hydroxycoumarin and 1-naphthol by rat liver slices were significantly decreased after cryopreservation in 12% DMSO, but they were maintained when the concentration of this cryopreservant was increased to 18%. 5. After cryopreservation in 12% DMSO, the mitochondrial reduction of the tetrazolium salt MTT by rat liver slices was significantly lowered. In contrast, slices cryopreserved in 18% DMSO demonstrated no significant decrease in their capacity to reduce MTT. 6. Thus, in agreement with previous studies, it was found that cytochrome P450-dependent activities are retained after cryopreservation of liver slices. Although phase II conjugating enzyme activities are more sensitive to cryopreservation, it was shown that increasing the concentration of DMSO present during cryopreservation could circumvent the problem. This modification improves the usefulness of cryopreserved rat liver slices as a tool in drug metabolism studies.     

Comparison of in vitro preparations for semi-quantitative prediction of in vivo drug metabolism.

Various in vitro preparations were compared with respect to their ability to mimic in vivo metabolism. For this purpose, S9-liver homogenate, microsomes, cryopreserved hepatocytes, cryopreserved liver slices and fresh liver, lung, kidney, and intestinal slices were incubated with three drugs in development, which are metabolized in vivo by a wide range of biotransformation pathways. Metabolites were identified and quantified with liquid chromatography-mass spectometry/UV from the in vitro incubations and compared with metabolite patterns in feces, urine, and bile of dosed rats. In vitro systems with intact liver cells produced the same metabolites as the rat in vivo and are a valuable tool to study drug metabolism. Phase I metabolites were almost all conjugated in intact cells, whereas S9-homogenate only conjugated by sulfation and N-acetylation. Microsomes and S9-homogenate are useful to study phase I metabolism but not for the prediction of in vivo metabolism. Extra-hepatic organ slices did not form any metabolites that were not produced by liver cells, but the relative amounts of the various metabolites differed considerably. Small intestinal slices were more active than liver slices in the formation of the N-glucuronide of compound C, which is the major metabolite in vivo. When the relative contribution of liver and small intestinal slices to the metabolism of this compound was taken into account, it appeared that the in vivo metabolite pattern could be well predicted. Results indicate that for adequate prediction of in vivo metabolism, fresh or cryopreserved liver slices or hepatocytes in combination with slices of the small intestines should be used.     

In vitro induction of cytochrome P450 2B1- and 3A1-mRNA and enzyme immunostaining in cryopreserved precision-cut rat liver slices

With the exception of cytochrome P450 (CYP) 1A1 and its mRNA, in vitro induction of other CYP forms has not been demonstrated in cryopreserved liver slices until now. Therefore precision-cut rat liver slices were cultured after cryopreservation and thawing in William's medium E for up to 24 h in the presence of inducers to demonstrate CYP2B1- and CYP3A1-mRNA induction. CYP-mRNA expression was determined by competitive RT-PCR. Exposure to 100 microM phenobarbital caused a more than 20-fold increase in CYP2B1-mRNA expression within 24 h, reaching concentrations comparable with those of PB-exposed fresh rat liver slices. Exposure to 1 microM pregnenolone 16 alpha-carbonitrile enhanced CYP3A1-mRNA expression by more than 30-fold within 24 h. This is in the same range, although with higher variability, as detected with fresh liver slices. In spite of considerable variability among the thawed slices, the induction factors are high enough for a sensitive detection of an induction at mRNA level. Additionally, immunostaining of respective CYP-forms was performed in sections of few samples, indicating CYP increase in viable cells of cryopreserved slices.     

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 hepatocytes are (1) the freezing protocol, (2) the concentration of the cryoprotectant [10% dimethyl-sulfoxide (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 > or = 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 > or = 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.     

Rates of metabolism of chlorzoxazone, dextromethorphan, 7-ethoxycoumarin, imipramine, quinidine, testosterone and verapamil by fresh and cryopreserved rat liver slices, and some comparisons with microsomes.

In the present study we have investigated the disappearance of chlorzoxazone, dextromethorphan, 7-ethoxycoumarin, imipramine, quinidine, testosterone and verapamil from the medium in which fresh and cryopreserved rat liver slices were incubated. These compounds are all substrates of major isoforms of cytochrome P450 expressed in the liver. The metabolism of five of these compounds in microsomes from rat liver was also examined. Determinations of the concentrations of the compounds were performed employing LC/MS. Intrinsic clearance values (CL(ints)) were calculated on the basis of the concentration-vs.-time curves. No significant differences in the CL(int) values obtained with fresh and cryopreserved rat liver slices were observed for any of the compounds. The highest CL(int) value estimated with liver slices was observed for testosterone and the lowest values were with chlorzoxazone and 7-ethoxycoumarin. The total CL(int) values for 7-ethoxycoumarin and imipramine, calculated using scaling factors, were similar for liver slices and microsomes. In the case of testosterone, this total CL(int) was approximately 3.7-fold lower, whereas for dextromethorphan and quinidine it was 2.5- and 8.5-fold higher, respectively, with liver slices than with microromes. In conclusion, the rate of metabolism of the seven compounds tested with rat liver slices was not affected by cryopreservation. This finding adds further support to the general conclusion that the major activities involved in drug metabolism are not affected by cryopreservation of rat liver slices.     

Characterization of testosterone metabolism and 7-hydroxycoumarin conjugation by rat and human liver slices after storage in liquid nitrogen for 1 h up to 6 months

1. Slices of human and rat liver were cryopreserved in 18% dimethyl sulphoxide (DMSO) and subsequently stored in liquid nitrogen for periods up to as long as 6 months. After thawing, the metabolism of testosterone to hydroxylated products and conjugation of 7-hydroxycoumarin were investigated. 2. Rat liver slices stored in liquid nitrogen for 6 months exhibited rates of formation of 7alpha-, 6beta- 16alpha- and 2alpha-hydroxytestosterone, and of androstenedione that did not differ significantly from those observed with fresh slices. 3. No formation of 2alpha-hydroxytestosterone was detected with slices of human liver. However, in contrast with the rat, human slices produced 2beta-hydroxytestosterone. The rates of formation of 7alpha-, 6beta-, 16alpha- and 2beta-hydroxytestosterone and of androstenedione by human liver slices after 6 months of storage in liquid nitrogen were 82, 71, 236, 66 and 92%, respectively, of the corresponding rates by fresh slices. 4. The rates of sulphation and glucuronidation of 7-hydroxycoumarin by slices from rat liver were 97 and 119%, respectively, of the corresponding fresh values after 6 months of storage in liquid nitrogen. 5. 7-Hydroxycoumarin glucuronidation by human liver slices was 53% of the corresponding fresh values after 6 months of storage. However, human slices showed little or no capacity to conjugate 7-hydroxycoumarin with sulphate. 6. It was demonstrated that slices of both human and rat liver can be cryopreserved and stored in liquid nitrogen for at least 6 months without major changes in their rates of metabolism of testosterone to its hydroxylated products and of 7-hydroxycoumarin conjugation. These findings further emphasize that cryopreservation of liver slices can be an effective tool in the use of biological material of limited availability.     

Optimization of cryopreservation procedures for rat and human hepatocytes

1. Rat hepatocytes were cryopreserved using a number of procedures and the viability, attachment, and metabolic activity of the cryopreserved cells were compared to freshly isolated hepatocytes. Several cryopreservation agents (dimethylsulphoxide [DMSO], glycerol, polyvinylpyrrolidone [PVP], dextrans), and combinations of these agents, were examined. Other variables tested included the freezing rate, thawing rate, and the concentration of serum in the freezing medium. 2. Recovery of viable attached cells was optimal using DMSO at concentrations of 10% or higher, a slow stepwise cooling procedure, and a quick thaw. The concentration of serum in the freezing medium (0% to 90%) did not affect cryopreservation results. Using this procedure the recovery of viable hepatocytes was 70%. 3. Levels of hepatocyte ethoxycoumarin-O-deethylase (ECOD) activity did not change following cryopreservation. The rate of decline of ECOD activity with time in culture was similar in freshly isolated and cryopreserved hepatocytes. 4. Hepatocytes isolated from three human livers were cryopreserved and recovered with viabilities similar to those obtained with the rat. A preliminary experiment also showed no loss of metabolic activity in human hepatocytes following cryopreservation.     

Xenobiotic metabolizing enzyme activities in isolated and cryopreserved human liver parenchymal cells

Liver parenchymal cells (hepatocytes) of human organ donors were isolated using a two-step collagenase perfusion technique. The average viability of the freshly isolated liver parenchymal cells, as judged by trypan blue exclusion, was 82% (SD = 7%; n = 6). The inter-individual differences in the determined enzyme activities were less than a factor of 7.5, despite the different sexes and ages of the donors. Freshly isolated parenchymal cells (PC) were cryopreserved using a computer-controlled freezing protocol. After thawing, cryopreserved cells had a mean viability of 57% (SD = 18%; n = 6). The activities of xenobiotic metabolizing enzymes in freshly isolated and cryopreserved cells were compared using PC from two donors. The enzyme activities of phenol sulfotransferase, 1-naphthol UDP-glucuronosyltransferase and microsomal epoxide hydrolase were well maintained after thawing (87–117% of activities in freshly isolated cells), whereas the activities of glutathione S-transferase, monitored with the broad spectrum substrate 1-chloro-2,4-dinitrobenzene, and the major broad spectrum cytosolic epoxide hydrolase were moderately but markedly reduced after cryopreservation (34–64% and 45–89% of levels in fresh cells, respectively). The decrease of both activities was dependent on the viability after thawing. When cryopreserved cells were purified by a Percoll centrifugation after thawing, the viability was increased from 62 to 92% for cells from one of the donors and from 88 to 98% for PC for the other donor. Subsequently the activity of glutathione S-transferase in Percoll-purified PC from the two donors was increased to 71 and 96% of levels in freshly isolated cells. It is concluded that the use of cryopreserved liver parenchymal cells of humans and other species represents a valuable tool in predicting which animal species best represents humans in hepatic metabolism and therefore should be the preferred species for investigations of metabolism and metabolism-dependent toxicities.     

Cryopreservation of rat and dog hepatocytes for studies of xenobiotic metabolism and activation

Isolated human hepatocytes offer a unique way of studying the metabolism and mechanisms of action of drugs and toxic chemicals. Because of the irregular availability of human liver, a way of storing the hepatocytes until they can be conveniently used is required. Using rat and dog isolated hepatocytes, we have developed a procedure for cryopreserving hepatocytes in large numbers such as are needed for metabolism and toxicity studies. Hepatocytes were frozen in medium containing 10% dimethyl sulfoxide using a microcomputer-controlled freezing gradient and stored at -196 degrees C. Upon thawing, the total cell recovery for rat hepatocytes was 67%. Cell viability measured by trypan blue (TB) exclusion was 67%, 7-ethoxycoumarin (7-EOC) dealkylation 33%, and cytochrome P-450 75%, compared to fresh hepatocytes. With cryopreserved dog hepatocytes, the total cell recovery was 75%. TB exclusion was 62%, 7-EOC dealkylation 37%, and cytochrome P-450 68%, compared to fresh hepatocytes. The viability of cryopreserved hepatocyte preparations could be improved by density separation on Percoll giving a TB exclusion for rat hepatocytes of 85%, and 7-EOC dealkylation of 69% compared to fresh hepatocytes, with 67% of the viable cells recovered. Biphenyl was used as a substrate to measure integrated xenobiotic metabolizing activity by the hepatocytes. Total hydroxybiphenyl (OHB) formation, a mixed function oxygenase activity, was maintained in cryopreserved Percoll-treated rat hepatocytes at 86%, OHB glucuronide formation at 85%, and OHB sulfate formation at 20% of the values in fresh hepatocytes. In cryopreserved dog hepatocyte, total OHB formation was 39%, and OHB glucuronide and sulfate formation less than 10% of the values in fresh hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of cryopreserved rat liver parenchymal cells by metabolism of diagnostic substrates and activities of related enzymes.

The metabolism of testosterone and benzo(a)pyrene (BaP) which is mediated by diverse enzymes was determined in cryopreserved rat liver parenchymal cells and compared with that found in freshly isolated cells. In addition, the activities of single xenobiotic-metabolizing enzymes were measured by using specific substrates. The cytochrome P450 (P450)-mediated total metabolic conversion of testosterone was reduced to 55% in cryopreserved cells. The metabolite profile, i.e. the formation of single metabolites compared with total metabolic conversion, was however unchanged when compared with freshly isolated cells. A concomitant reduction in the activities of the involved P450 isoenzymes can therefore be postulated. The amount of detected phase I-metabolites of BaP was unaffected by the cryopreservation method. The formation of phase II-metabolites and total metabolic conversion of BaP in cryopreserved cells was however reduced to about 50-60%. The reduced glutathione S-transferase and more obviously phenol sulfotransferase activities measured in cryopreserved cells, may explain the impaired conjugation of BaP. The ratio between phase I- and phase II-metabolites was thus changed by cryopreservation. Density separation on Percoll yielded cryopreserved cells with a viability and metabolic capacity not measurably different from freshly isolated cells. To this extent, cryopreserved, Percoll-purified liver parenchymal cells are a useful in vitro system for drug metabolism studies. However due to the extensive loss in cell number during this procedure (recovery = 22% of freshly isolated cells) the application of this system is limited.     

Biotransformation of halothane in guinea pig liver slices

The biotransformation of halothane was studied using liver slices. Precision-cut Hartley male guinea pig liver slices (1 cm diameter; 250-300 microns thick) were incubated in sealed roller vials containing supplemented Krebs-Henseleit buffer at 37 degrees C under different O2 tensions (2.5, 21, and 95%). After a 1-hr preincubation, halothane was vaporized in the vial producing a 1.9 mM medium concentration. Halothane metabolites (Br-, trifluoroacetic acid, F-) were measured at 2, 4, and 6 hr. Viability of the incubated slices was verified by determining intracellular K+ content and levels of cytochrome P-450, which were maintained under 95% O2 atmosphere but decreased with lower O2 tensions (2.5%). The highest fluoride production was 300 +/- 22 pmol/mg slice weight/6 hr at low O2 tension (2.5%). Defluorination decreased with increasing O2 tension to undetectable levels under 95% O2. Production of the oxidative metabolite, trifluoroacetic acid, was highest at 95% O2 (2.35 +/- 0.17 nmol/mg slice weight/6 hr). Trifluoroacetic acid production decreased with decreasing O2 tension. Br- production was the highest at 21% O2 (1.8 +/- 0.13 nmol/mg slice weight/6 hr). Production of Br- was not dependent on the O2 tension. The guinea pig slices are capable of biotransforming halothane (oxidative/reductive); therefore, this in vitro system appears suitable for studying the biotransformation of halothane.     

Impaired glutathione-conjugating capacity by cryopreserved human and rat hepatocytes

The activity of glutathione transferase was measured in sonicates of fresh rat hepatocytes and of cryopreserved rat, human and dog hepatocytes in the presence of added glutathione and by using 1-chloro-2,4-dinitrobenzene (CDNB) as non-selective substrate. The glutathione-conjugating capacity was also investigated in the presence of CDNB alone (without glutathione) with intact fresh rat hepatocytes and cryopreserved rat and human hepatocytes. Finally, the intracellular level of glutathione was measured in these hepatocytes. The specific activity of glutathione transferase in sonicates of fresh rat hepatocytes (in the presence of added GSH and CDNB) was about 415 nmol/min/10(6) cells. The corresponding activities in cryopreserved rat, human and dog hepatocytes were approximately 320, 440 and 540 nmol/min/10(6) cells, respectively. In contrast, glutathione conjugation by the intact cryopreserved human and rat hepatocytes in the presence of CDNB alone was less than 10% of the corresponding conjugation by fresh rat hepatocytes, indicating that glutathione was depleted in these cryopreserved hepatocytes. Glutathione depletion was confirmed after analytical measurement of the glutathione levels in fresh and cryopreserved hepatocytes. In fresh rat hepatocytes the level of glutathione was 44 nmol/10(6) cells, whereas it was 2.5 and 4.4 nmol/10(6) cells in cryopreserved rat and human hepatocytes, respectively. In summary, glutathione transferase was active in these cryopreserved hepatocytes but the cryopreservation procedure likely causes depletion in the intracellular level of glutathione, resulting in an overall reduced glutathione conjugating capacity.     

Optimizing preincubation conditions for precision-cut rat kidney and liver tissue slices: effect of culture media and antioxidants

Tissue slices are commonly ‘preincubated’ before use, but optimal conditions to ensure their maximal viability have not been systematically investigated. The effects of serum-free Dulbecco's minimum Eagle's medium and Ham's nutrient mixture (DMEM/F12) (1:1) culture media with and without phenol red (±PR), or RPMI-1640 and six different antioxidants on the viability of precision-cut rat kidney and liver slices (200±5 μm) were investigated. Slice viability was assessed every 30 minutes over a 2-hour preincubation period and after 24 hours of incubation in a multiwell plate culture system maintained at 37°C. In all cases, preincubation produced a time-dependent significant reduction of ethidium bromide positive nuclei stained in each medium and in both kidney and liver slices. Based on lactate dehydrogenase (LDH) leakage, there are viability differences between the media. In contrast, alkaline phosphatase (ALP) leakage and MTT reduction were less sensitive and did not differentiate between slice viability in each incubation medium. Preincubation of kidney and liver slices in DMEM/F12 medium containing antioxidants, indicated an enhanced viability which was specific for each tissue. Extension of the culture period to 24 hours after 1 hour of preincubation showed up to a further 4–13% leakage of ALP or LDH in DMEM/F12 (±PR) media for both kidney and liver slices and with a further 5–15% decline in MTT viability assay. RPMI-1640 medium on its own was not a suitable medium for maintaining the viability of either kidney or liver slices. However, kidney or liver slices preincubated with DMEM/F12 medium in the presence of some of the antioxidants were satisfactorily maintained for 24 hours. Exposure of slices to atractyloside (ATR) at concentrations of 0.2–2.0 m in the different media for 24 hours showed a significant increase in enzyme leakage, decline of MTT reductive capacity and increased oxidative damage, with toxicity more elaborate in RPMI-1640 medium. Preincubation of kidney slices with either reduced glutathione (GSH) or -tocopherol (TOC) and liver slices with either GSH or deferoxamine (DEF) followed by 24 hours of exposure to ATR showed a similar decline in toxicity profile. The antioxidants provided partial protection of slices from ATR toxicity. The results demonstrate the importance of slice preincubation and indicate that slices could be maintained in culture using an appropriate medium, thus providing slices that could serve as a useful alternative in vitro system for assessing novel compounds for toxicity.     

Characterization of testosterone metabolism and 7-hydroxycoumarin conjugation by rat and human liver slices after storage in liquid nitrogen for 1 h up to 6 months

1. Slices of human and rat liver were cryopreserved in 18% dimethyl sulphoxide (DMSO) and subsequently stored in liquid nitrogen for periods up to as long as 6 months. After thawing, the metabolism of testosterone to hydroxylated products and conjugation of 7-hydroxycoumarin were investigated. 2. Rat liver slices stored in liquid nitrogen for 6 months exhibited rates of formation of 7α-, 6β- 16α- and 2β-hydroxytestosterone, and of androstenedione that did not differ significantly from those observed with fresh slices. 3. No formation of 2β-hydroxytestosterone was detected with slices of human liver. However, in contrast with the rat, human slices produced 2β-hydroxytestosterone. The rates of formation of 7α-, 6β-, 16α- and 2β-hydroxytestosterone and of androstenedione by human liver slices after 6 months of storage in liquid nitrogen were 82, 71, 236, 66 and 92%, respectively, of the corresponding rates by fresh slices. 4. The rates of sulphation and glucuronidation of 7-hydroxycoumarin by slices from rat liver were 97 and 119%, respectively, of the corresponding fresh values after 6 months of storage in liquid nitrogen. 5. 7-Hydroxycoumarin glucuronidation by human liver slices was 53% of the corresponding fresh values after 6 months of storage. However, human slices showed little or no capacity to conjugate 7-hydroxycoumarin with sulphate. 6. It was demonstrated that slices of both human and rat liver can be cryopreserved and stored in liquid nitrogen for at least 6 months without major changes in their rates of metabolism of testosterone to its hydroxylated products and of 7-hydroxycoumarin conjugation. These findings further emphasize that cryopreservation of liver slices can be an effective tool in the use of biological material of limited availability.     

微囊化牛嗜铬细胞及冷冻保存的体外培养研究

目的 观察体外培养的微囊化牛嗜铬细胞及其冷冻保存复苏后的细胞活力,探索微囊化牛嗜铬细胞的冻存保存方法,为将来进行大规模细胞移植治疗疼痛奠定临床基础.方法 经胶原酶消化成单细胞,微囊包裹,并取部分进行冷冻保存及复温,观察细胞的生长情况;放免法分别检测其培养液中去甲肾上腺素、甲-脑啡呔的含量和在高钾、乙酰胆碱刺激下的分泌量来观察其功能活性.结果 微囊化牛嗜铬细胞及其冷冻复苏后的细胞形态结构完整,生长良好;冻存复苏后的微囊保留了儿茶酚胺和甲-脑啡呔的的分泌功能,基础与刺激分泌量大约为冻存前微囊化牛嗜铬细胞分泌量的92%;同时刺激后去甲肾上腺素、甲-脑啡呔水平较其基础值均有明显增加（P＜0.01）.结论 微囊化牛嗜铬细胞及其冷冻复苏后的细胞具有良好的细胞功能,冷冻保存的微囊化牛嗜铬细胞是有效和成功的.