Cryopreservation of rat precision-cut liver and kidney slices by rapid freezing and vitrification

Precision-cut tissue slices of both hepatic and extra-hepatic origin are extensively used as an in vitro model to predict in vivo drug metabolism and toxicity. Cryopreservation would greatly facilitate their use. In the present study, we aimed to improve (1) rapid freezing and warming (200 degrees C/min) using 18% Me(2)SO as cryoprotectant and (2) vitrification with high molarity mixtures of cryoprotectants, VM3 and VS4, as methods to cryopreserve precision-cut rat liver and kidney slices. Viability after cryopreservation and subsequent 3-4h of incubation at 37 degrees C was determined by measuring ATP content and by microscopical evaluation of histological integrity. Confirming earlier studies, viability of rat liver slices was maintained at high levels by rapid freezing and thawing with 18% Me(2)SO. However, vitrification of liver slices with VS4 resulted in cryopreservation damage despite the fact that cryoprotectant toxicity was low, no ice was formed during cooling and devitrification was prevented. Viability of liver slices was not improved by using VM3 for vitrification. Kidney slices were found not to survive cryopreservation by rapid freezing. In contrast, viability of renal medullary slices was almost completely maintained after vitrification with VS4, however vitrification of renal cortex slices with VS4 was not successful, partly due to cryoprotectant toxicity. Both kidney cortex and medullary slices were vitrified successfully with VM3 (maintaining viability at 50-80% of fresh slice levels), using an optimised pre-incubation protocol and cooling and warming rates that prevented both visible ice-formation and cracking of the formed glass. In conclusion, vitrification is a promising approach to cryopreserve precision-cut (kidney) slices.     

Cryopreservation of rat and human liver slices by rapid freezing

The cryopreservation of human liver slices is a promising way to enhance the ability to test the metabolism of drug candidates. This study demonstrates the use of a novel technique for the cryopreservation of both rat and human liver slices. In this technique the slices are treated with Me2SO and sandwiched between aluminum plates separated by a thin gasket. The device is then submerged in liquid nitrogen to freeze the slices, which can then be stored until use. To thaw the slices, the apparatus is submerged in a water bath at 37 degrees C. Slices frozen and thawed in this manner were compared to those frozen in conventional cryovials. The viability of the slices was determined by incubating them in 12-well plates and measuring urea synthesis, ethoxycoumarin metabolism, and cytosolic enzyme leakage (LDH and ALT). The viability of rat slices frozen between plates approached that of fresh slices and was consistently higher than slices frozen in cryovials. Slices from two human samples gave similar results. The technique was found to work over a wide range of Me2SO concentrations (4.5 to 22% was tested) with an optimal concentration between 10 and 15%.     

Cryopreserved human thyroid tissue for TSAb determination: comparison with fresh tissue

A thyroid-stimulating antibody assay using human normal thyroid slices was performed for studying whether the cryopreservation technique produced a sensitive substrate for cyclic-AMP generation. Cryopreservation was carried out in sterile 15% glycerol 0.9% NaCl placed in a hexane-nitrogen bath at -196 degrees C and stored at -80 degrees C for 1-5 weeks. Dose-response curves for untreated Graves' hyperthyroid patients IgG and bovine TSH were similar using fresh and cryopreserved slices. Assay sensibility was 5 mU/ml TSH. A high correlation (r = 0.91; p less than 0.001; n = 41) was found for percentage of cyclic-AMP increase to b-TSH and Graves' IgG comparing both methods. Studying 19 Graves' hyperthyroid patients, positive results were found in 63 and 52% for fresh and cryopreserved slices respectively. The cryopreservation technique used in the study was efficient for TSAb determinations.     

A new approach to the cryopreservation of hepatocytes in a sandwich culture configuration

Current methods of cryopreservation of hepatocytes in single cell suspensions result in low overall yields of hepatocytes, demonstrating long-term preservation of hepatocellular functions. A novel culture method has recently been developed to culture liver cells in a sandwich configuration of collagen layers in order to stabilize the phenotypic expression of these cells in vitro (J. C. Y. Dunn, M. L. Yarmush, H. G. Koebe, and R. G. Tompkins, FASEB J. 3, 174, 1989). Using this culture system, rat hepatocytes were frozen with 15% (v/v) Me2SO to -70 degrees C, and stored at approximately -100 degrees C. Following rapid thawing, long-term function was assessed by measuring albumin secretion in culture for 7-14 days postfreezing. Comparison was made with cryopreservation of liver cells in single cell suspensions. Cryopreservation of liver cells in suspension resulted in only a 2% yield of cells which could be successfully cultured; albumin secretion rates in these cultured cells over 48 hr were 26-30% of secretion rates for nonfrozen hepatocytes. Freezing cultured liver cells in the sandwich configuration after 3, 7, and 11 days in culture maintained 0, 26, and 19% of the secretion rates of nonfrozen hepatocytes, respectively. Morphology of the cryopreserved cells appeared grossly similar to cells without freezing; however, this morphological result was patchy and represented approximately 30% of the cells in culture. These results represent the first demonstration of any quantitative long-term preservation of hepatocellular function by cryopreservation, suggesting that cultured hepatocytes can survive freezing and maintain function.     

Method of preparing suspended cells for scanning electron microscopy]

To preserve the lifetime morphology of the surface of suspended cells, these must be fixed in suspensions. The subsequent stages of cell preparation for scanning electron microscopy (dehydratation, critical point drying, coating) are considerably facilitated if fixed cells are preliminary attached to some substrate surface. An effective substrate should provide a firm rather than selective attachment of the overwhelming majority of fixed cells; the substrate should be also available for a wide application. The trial of different types of substrates showed a sufficient effectivity of plates made of commercial aluminium foil. In tests with murine embryonal and transformed fibroblasts as well as with human blood leukocytes, in average 90 per cent of cells fixed with glutaraldehyde in suspensions got attached to foil substrate surfaces; the fixed cells both settled from suspension and attached were seen distributed evenly on the substrate surface. The use of aluminium foil substrates made it possible to study the surface topography of some types of suspended cells.     

Efficient analysis of hepatic glucose output and insulin action using a liver slice culture system.

BACKGROUND: Liver slices have been reported to retain histological integrity and metabolic capacity for over 24 hours in flask culture systems, and they have been used for pharmacological and toxicological studies before. However, whether this method is suitable to measure hepatic glucose output is unknown. METHODS: Precision-cut liver slices were prepared from fresh male rat liver. After high-glucose pre-incubation (11.2 mmol/l), medium was changed to low-glucose conditions (0.5 mmol/l). Glucose and lactate levels as well as aspartate aminotransferase activity were monitored for 50 minutes with or without addition of insulin (600 pmol/l) and/or epinephrine (0.5 micromol/l). Slice potassium content and histology were examined to prove liver viability. RESULTS: We observed a stable glucose production from the liver slices of 0.3-0.4 micromol/g liver/min. Epinephrine increased (by 82+/-30%) and insulin decreased (by 80+/-8%) liver slice glucose output. Significant signs of ischemia were not detected. CONCLUSIONS: Hepatic glucose release can be reliably measured in a liver slice culture system, and it is regulated by major hormone systems. This method may be helpful for further characterization of direct insulin action and resistance in a complex tissue as the liver; however, pharmacological applications such as the analysis of drug effects on hepatic glucose metabolism can also be envisioned.     

Opposing changes in 3alpha-hydroxysteroid dehydrogenase oxidative and reductive activities in rat leydig cells during pubertal development

The enzyme 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) has an important role in androgen metabolism, catalyzing the interconversion of dihydrotestosterone (DHT) and 5alpha-androstane-3alpha,17beta-diol (3alpha-DIOL). The net direction of this interconversion will affect the amount of biologically active ligand available for androgen receptor binding. We hypothesize that in Leydig cells, differential expression of 3alpha-HSD enzymes favoring one of the two directions is a mechanism by which DHT levels are controlled. In order to characterize 3alpha-HSD in rat Leydig cells, the following properties were analyzed: rates of oxidation (3alpha-DIOL to DHT) and reduction (DHT to 3alpha-DIOL) and preference for the cofactors NADP(H) and NAD(H) (i.e., the oxidized and reduced forms of both pyridine nucleotides) in Leydig cells isolated on Days 21, 35, and 90 postpartum. Levels of 3alpha-HSD protein were measured by immunoblotting using an antibody directed against the liver type of the enzyme. Levels of 3alpha-HSD protein and rates of reduction were highest on Day 21 and lowest on Day 90. The opposite was true for the rate of 3alpha-HSD oxidation, which was barely detectable on Day 21 and highest on Day 90 (59.08 +/- 6.35 pmol/min per 10(6) cells, mean +/- SE). Therefore, the level of 3alpha-HSD protein detectable by liver enzyme was consistent with reduction but not with oxidation. There was a clear partitioning of NADP(H)-dependent activity into the cytosolic fraction of Leydig cells, whereas on Days 35 and 90, Leydig cells also contained a microsomal NAD(H)-activated 3alpha-HSD. We conclude that 1) the cytosolic 3alpha-HSD in Leydig cells on Day 21 behaves as a unidirectional NADPH-dependent reductase; 2) by Day 35, a microsomal NAD(H)-dependent enzyme activity is present and may account for predominance of 3alpha-HSD oxidation over reduction and the resultant high capacity of Leydig cells on Day 90 to synthesize DHT from 3alpha-DIOL.     

Apolipoprotein B production and very low density lipoprotein secretion by calf liver slices.

Secretion of triglycerides by the liver in ruminants as components of very low density lipoproteins particles is low as compared with that in primates or rodents. The rate-limiting steps for the hepatic export of very low density lipoproteins have been studied in liver slices to determine the origin of the low lipotropic capacity of calf liver compared to that of rat liver. The rates of production of apolipoprotein B (apo B) and albumin as well as the rate of secretion of VLDL-apolipoproteins were measured during 12-h incubation of liver slices in organo-culture using [35S]methionine-cysteine labeling. Hepatic apo B production was similar in the two animal species but the VLDL-apolipoprotein secretion rate for calf liver slices amounted to only 20% of that observed for rat liver slices. Although calf and rat liver slices synthesized similar amounts of total protein, the hepatic production of albumin, measured in cells and media, was much higher in calf than rat liver slices (around 2.7-fold), whereas the rate secretion of albumin was similar in the two species. Our results showed that the slow rate of secretion of VLDL by calf liver cells was not consecutive to a low rate of synthesis of apo B but rather to a defect in VLDL assembly and/or secretion.     

Hydrogel embedding of precision-cut liver slices in a microfluidic device improves drug metabolic activity

A microfluidic-based biochip made of poly-(dimethylsiloxane) was recently reported for the first time by us for the incubation of precision-cut liver slices (PCLS). In this system, PCLS are continuously exposed to flow, to keep the incubation environment stable over time. Slice behavior in the biochip was compared with that of slices incubated in well plates, and verified for 24 h. The goal of the present study was to extend this incubation time. The viability and metabolic activity of precision-cut rat liver slices cultured in our novel microflow system was examined for 72 h. Slices were incubated for 1, 24, 48, and 72 h, and tested for viability (enzyme leakage (lactate dehydrogenase)) and metabolic activity (7-hydroxycoumarin (phase II) and 7-ethoxycoumarin (phase I and II)). Results show that liver slices retained a higher viability in the biochip when embedded in a hydrogel (Matrigel) over 72 h. This embedding prevented the slices from attaching to the upper polycarbonate surface in the microchamber, which occurred during prolonged (>24 h) incubation in the absence of hydrogel. Phase II metabolism was completely retained in hydrogel-embedded slices when medium supplemented with dexamethasone, insulin, and calf serum was used. However, phase I metabolism was significantly decreased with respect to the initial values in gel-embedded slices with medium supplements. Slices were still able to produce phase I metabolites after 72 h, but at only about ～10% of the initial value. The same decrease in metabolic rate was observed in slices incubated in well plates, indicating that this decrease is due to the slices and medium rather than the incubation system. In conclusion, the biochip model was significantly improved by embedding slices in Matrigel and using proper medium supplements. This is important for in vitro testing of drug metabolism, drug-drug interactions, and (chronic) toxicity.     

Separation and quantitative determination of dolichol and dolichyl phosphate in rat and trout liver

The dolichol concentrations in rat and trout liver were found respectively to be 50-59 and 16-21 micrograms/g using three experimental methods: densitometric scanning of thin-layer plates, colorimetric assay and HPLC analysis. By HPLC of benzoylated dolichols, the distribution of the dolichols according to the number of their isoprene residues, was determined in rat and trout liver. The major component was dolichol -18 in rat and dolichol -19 in trout liver. Dolichyl phosphate concentrations were found to be 6-7 micrograms/g of rat liver and 8-9 micrograms/g of trout liver by densitometric scanning of thin-layer plates.     

Use of precision cut human liver slices for studying the metabolism and genotoxic potential of xenobiotics by means of the 32P-postlabelling technique: steps towards method validation using testosterone and 2-aminofluorene

In the present study, a new in vitro model combining the short-term incubation of precision-cut human liver slices with DNA-adduct analysis by the ³²P-postlabelling technique is proposed for investigation of the genotoxic potential of xenobiotics. For method validation, the metabolic turnover of testosterone (TES) and the DNA-adduct inducing potential of 2-aminofluorene (2-AF) were used. Precision-cut human liver slices were prepared from a total of 12 human liver samples which were freshly obtained as parts of resectates from liver surgery. The slices were incubated as submersion cultures with TES and 2-AF for up to 6 h in 12-well tissue culture plates at concentrations of 10-50 and 0.06-28 μM, respectively. Slices recovered from the slicing procedure in the 4 °C cold Krebs-Henseleit buffer as indicated by intracellular potassium concentrations which increased for 2 h and then remained stable until the end of the incubation. TES was extensively metabolized by human liver slices with a similar metabolite pattern as observed in vivo. Almost 90% of the metabolites were conjugates. Major phase-I metabolites were androstendione, 6β-OH-androstendione, 6β-OH-TES, and 15β-OHTES. After incubation with 2-AF, substance related DNA-adducts were detected which increased dose-dependently from 12 to 1146 adducts per 10⁹ nucleotides. The adduct pattern consisted of one major adduct spot, A, representing 80-90% of the total adduct level and up to four minor adduct spots, B-E. In summary, the present data demonstrate that precision-cut liver slices are a valuable alternative in vitro system for DNA-adduct determination to screen chemicals for potential genotoxicity in humans.     

Use of precision cut human liver slices for studying the metabolism and genotoxic potential of xenobiotics by means of the 32P-postlabelling technique: steps towards method validation using testosterone and 2-aminofluorene

In the present study, a new in vitro model combining the short-term incubation of precision-cut human liver slices with DNA-adduct analysis by the ³²P-postlabelling technique is proposed for investigation of the genotoxic potential of xenobiotics. For method validation, the metabolic turnover of testosterone (TES) and the DNA-adduct inducing potential of 2-aminofluorene (2-AF) were used. Precision-cut human liver slices were prepared from a total of 12 human liver samples which were freshly obtained as parts of resectates from liver surgery. The slices were incubated as submersion cultures with TES and 2-AF for up to 6 h in 12-well tissue culture plates at concentrations of 10-50 and 0.06-28 μM, respectively. Slices recovered from the slicing procedure in the 4 °C cold Krebs-Henseleit buffer as indicated by intracellular potassium concentrations which increased for 2 h and then remained stable until the end of the incubation. TES was extensively metabolized by human liver slices with a similar metabolite pattern as observed in vivo. Almost 90% of the metabolites were conjugates. Major phase-I metabolites were androstendione, 6β-OH-androstendione, 6β-OH-TES, and 15β-OHTES. After incubation with 2-AF, substance related DNA-adducts were detected which increased dose-dependently from 12 to 1146 adducts per 10⁹ nucleotides. The adduct pattern consisted of one major adduct spot, A, representing 80-90% of the total adduct level and up to four minor adduct spots, B-E. In summary, the present data demonstrate that precision-cut liver slices are a valuable alternative in vitro system for DNA-adduct determination to screen chemicals for potential genotoxicity in humans.     

Experiences with the critical review process of aluminium LCI data

Background, aim, and scope  

This paper summarises the critical review process according to ISO 14040/44 performed for the European Aluminium Association (EAA), Brussels. Scope of the review was a life cycle inventory (LCI) project, aiming at providing the life cycle assessment (LCA) community with reliable generic data relevant for the European aluminium market, including the production of aluminium ingot either from primary aluminium or from recycled aluminium and the fabrication of semi-finished products, i.e. sheet, foil or extrusion fabrication from aluminium ingots.     

The cell-surface expression of the cell adhesion molecule cellCAM 105 in rat fetal tissues and regenerating liver

In the present investigation we have used a sensitive immunohistochemical technique to study the appearance and cell-surface distribution of cellCAM 105 in rat fetal tissues and in regenerating liver. CellCAM 105 is an integral membrane glycoprotein that is involved in cell-cell adhesion of mature rat hepatocytes in vitro. In 12-day-old rat fetuses no cellCAM 105 was detected. CellCAM 105 then appeared on Day 13 in megakaryocytes of the fetal liver, on Day 16 in the liver parenchyme, and on Day 17 in the epithelial cells of the proximal kidney tubules and of the small intestinal mucosa. In the liver parenchyme cellCAM 105 first appeared in immature bile canaliculi. During Days 19-21 a significant staining also occurred on the contiguous sides of the hepatocytes, which at that time became closely associated when the blood-forming cells disappeared. This surface staining then gradually disappeared and 2-3 weeks after birth cellCAM 105 was expressed in the bile canalicular area which is typical of mature hepatocytes. In regenerating liver the amount of cellCAM 105 decreases to a minimum 2-3 days post-hepatectomy, then increases and reaches the normal concentration 10-15 days post-hepatectomy [Odin and Obrink (1986) Exp. Cell Res. 164, 103-114]. The cell-surface distribution of cellCAM 105 also changed, and on Days 3-5 post-hepatectomy it appeared on all faces of the hepatocytes which then were closely associated without obvious sinusoids in between. This staining pattern then slowly changed toward the normal pattern of mature liver, which appeared about 15 days post-hepatectomy. A theoretical analysis of the mode of hepatocyte cell division during liver regeneration suggested that the surface of the postmitotic hepatocytes should become unpolarized with respect to macromolecular composition. This is in agreement with the observed surface distribution of cellCAM 105. The results support the hypothesis that cell-surface interactions mediated by cellCAM 105 might contribute to the regular organization of hepatocytes in the normal, mature liver plates.     

Aluminium foil as an alternative substrate for the spectroscopic interrogation of endometrial cancer

Biospectroscopy has the potential to investigate and characterize biological samples and could, therefore, be utilized to diagnose various diseases in a clinical environment. An important consideration in spectrochemical studies is the cost‐effectiveness of the substrate used to support the sample, as high expense would limit their translation into clinic. In this paper, the performance of low‐cost aluminium (Al) foil substrates was compared with the commonly used low‐emissivity (low‐E) slides. Attenuated total reflection‐Fourier transform infrared spectroscopy was used to analyse blood plasma and serum samples from women with endometrial cancer and healthy controls. The 2 populations were differentiated using principal component analysis with support vector machines with 100% sensitivity in plasma samples (endometrial cancer = 70; healthy controls = 15) using both Al foil and low‐E slides as substrates. The same sensitivity results (100%) were achieved for serum samples (endometrial cancer = 60; healthy controls = 15). Specificity was found higher using Al foil (90%) in comparison to low‐E slides (85%) and lower using Al foil (70%) in comparison to low‐E slides in serum samples. The establishment of Al foil as low‐cost and highly performing substrate would pave the way for large‐scale, multicentre studies and potentially for routine clinical use.

     

Optimum conditions for detecting hepatic micronuclei caused by numerical chromosome aberration inducers in mice

To ascertain an optimum condition for detecting micronuclei in the liver caused by numerical aberration inducers, either carbendazim (125-1000mg/kg, p.o.), colchicine (0.375-1.5mg/kg, i.v.), cytochalasin B (2.5-20mg/kg, i.v.), diazepam (3.13-25mg/kg, i.v.), noscapine (7.8-62.5mg/kg, i.v.), paclitaxel (1-100mg/kg, i.v.) or trichlorfon (18.75-150mg/kg, i.v.) was administered once to male Slc:ddY mice 1 day before or after partial hepatectomy (PH, Day 1). Five days after PH (on Day 6), hepatic micronuclei were determined in conjunction with classifications of the main nuclei and relative liver weights as a proliferative indicator or a dysfunction marker of cell division. Additionally, hepatocyte proliferation index (HPI) was calculated by using mono-, bi- and multinucleated cell counts. Treatment of mice with six compounds, except for colchicine, after PH showed higher incidence of micronucleated hepatocytes (MNH) than that before PH, and also increases in binucleated and multinucleated cells. Especially for carbendazim, diazepam, noscapine and trichlorfon, the dosing after PH was essential for the detecting numerical aberration. Colchicine evidently increased HPI and decreased relative liver weights without MNH induction on Day 6. On Day 8 when HPI and relative liver weights almost returned to the basal range, a significant increase in MNH was noted. This implied that the strong inhibition of colchicine on hepatocyte proliferation may obstruct the induction of MNH on Day 6. In conclusion, to detect the potential numerical aberration, exposure of mice to test chemicals should be performed 1 day after PH, during which enhanced proliferation of hepatocytes was seen, and it would be better to analyze the liver specimens on Day 6 or more post-PH.     

Steroidogenic capacity and ultrastructural morphology of cultured ovine luteal cells

Corpora lutea were surgically collected from superovulated ewes 36 h post-injection of human chorionic gonadotropin (hCG) (Day 2), dissociated (0.2% collagenase), plated, and maintained in culture Days 2-10 in Medium 199 supplemented with 5% calf serum. Accumulation of progesterone in the cultures did not decrease (p greater than 0.05) from Day 3 (17.5 +/- 5.1 nmol/10(6) cells) to Day 10 (4.8 +/- 1.7 nmol/10(6) cells). Calf serum (5%) in the medium supported greater (p less than 0.05) progesterone production than fetal calf serum (5%) or medium without added serum. Steroidogenic cells did not increase (Days 2-10) in numbers, but increased (p less than 0.01) in mean cell diameter (Day 2, 11.7 +/- 0.4 micron; Day 10, 24.5 +/- 1.6 micron). Steroidogenic capacity on Day 10 of cells cultured Days 2-10 (in vitro) was not different (p greater than 0.05) from that of cells collected from the ovary on Day 10 (in vivo); however, steroidogenic cells recovered from plates had greater (p less than 0.01) mean cell diameters (24.5 +/- 1.6 micron, in vitro, compared to 15.2 +/- 1.0 micron, in vivo). Transmission electron microscopy revealed that cultured cells (Days 5, 10) possessed less smooth endoplasmic reticulum but more lipid droplet inclusions, ribosomes, and rough endoplasmic reticulum than cells obtained in situ (Day 10). Electron-dense secretory granules were rarely seen. Although subcellular morphology of ovine luteal cells in culture was altered, these changes did not appear to significantly affect the ability of these cells to produce progesterone.     

Specific PGF-2 alpha binding by the corpus luteum of the pregnant and non-pregnant mare.

The binding of prostaglandin (PG) F-2 alpha to corpora lutea (CL) from pregnant and non-pregnant Pony mares was examined. Studies of the rates of association and dissociation indicated that [3H]PGF was bound specifically and reversibly to a luteal cell membrane preparation (MP) that was isolated by high speed (100,000 g) ultracentrifugation. Various PGs and PG metabolites displaced [3H]PGF from the receptors in the following decreasing order: PGF-2 alpha greater than 13, 14-dihydro-PGF-2 alpha = 13,14-dihydro-15-keto PGF-2 alpha greater than PGD-2 greater than PGF-1 alpha = PGE-2 greater than PGE-2 beta greater than PGE-1. These data implicate the 9 alpha-OH and 5,6 cis double bond as major contributors to PGF receptor recognition. The membrane preparation appeared to contain at least two receptor populations, a high affinity, low capacity and a low affinity, high capacity receptor. The binding of PGF (pg/mg MP protein +/- s.e.m. (n)) to CL of the non-pregnant mare increased from 4.09 +/- 11.6 (4), on Day 4 after ovulation, to reach maximal levels by Day 12, 15.01 +/- 2.5 (4), and declined thereafter. In pregnancy the binding of PGF continued to increase until Day 18, reaching 27.47 +/- 1.7 (3), before it declined on Day 20. The reduction in binding by Day 16 in the non-pregnant mare may reflect the process of luteolysis, while high PGF binding capacity of CL between Days 16 and 18 of pregnancy indicated that luteal maintenance during pregnancy is not associated with a reduction of PGF binding capabilities.     

A mechanical device for the rapid removal and freezing of liver or brain tissue from unanaesthetized and nonparalyzed rats.

A mechanical, pneumatically operated device has been developed that permits withdrawal and freezeclamping of either liver or brain tissue from unanaesthetized nonimmobilized rats. Furthermore, regional sampling from brain is possible. The apparatus works in the following way: By means of rotating knives, two cross-sections are made through a rat in such a way that the slice cut out of the animal between the two knives will contain the desired tissue. This slice is then freezeclamped between precooled aluminium blocks. The sampling procedure works automatically: i.e., when the rat is in position in the apparatus and the precooled aluminium blocks have been mounted on the arms of a pair of cooling tongs, the biopsy is made simply by pressing a button. The time for making a biopsy amounts to 0.10 sec. The brain or liver tissue can be isolated from the freezeclamped slices at the temperature of liquid N₂, using a small buzz saw developed for this purpose.     

Reoxygenation after cold hypoxic storage of cultured precision-cut rat liver slices: effects on cellular metabolism and drug biotransformation.

Cultured rat precision-cut liver slices (PCLS) were used to study the influence of hypothermic preservation and reoxygenation at 37 degrees C on cellular metabolism and drug biotransformation. Cold hypoxic storage caused a depressed metabolism in rat liver slices, but reoxygenation for 8 h at 37 degrees C partially restored the levels of both ATP and GSH and totally restored the capacity to synthesize proteins. Metabolism of midazolam (CYP3A-dependent oxidation) by cold preserved liver slices was decreased by 30% but no further affected by reoxygenation, showing the same profile as freshly cut slices. Such a reoxygenation at 37 degrees C is accompanied by a dramatic loss of CYP3A2 protein while CYP3A1 protein was unaffected. These results suggest that CYP3A2 did not play a major role in midazolam oxidation. Such results are not consistent with a putative reoxygenation injury but rather with cold hypoxic damage. Since cold preserved liver slices did not respond to bacterial endotoxin stimulation (lipopolysaccharides), a minor role of non-parenchymal cells is suggested as mediators for deleterious effects developed during the cold storage.