Assessment of Some Critical Factors in the Freezing Technique for the Cryopreservation of Precision-Cut Rat Liver Slices

Maas, Wilfred; Graaf, Inge A. M. de; Schoen, Eric D.; Koster, Henk; Sandt, J.J.M. van de; Groten, J.P.

doi:10.1006/cryo.2000.2246

Cited by 38 publications

(19 citation statements)

References 17 publications

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“…Only few studies report successful cryopreservation of PCLS and most demonstrate that only few functions such as CYP-dependent oxidative metabolism are maintained for several hours upon incubation after thawing, whereas highly "energy" consuming processes such as drug conjugation, or lipid and protein synthesis -as confirmed in the present study -are rapidly lost upon incubation [3,[8][9][10].…”

Section: Discussionmentioning

confidence: 54%

“…Several PCLS cryopreservation methods, including slow or fast freezing and vitrification methods, have been described in recent years (for an overview see [2]). Most of the data concern the use of cryopreserved PCLS in xenobiotics metabolism: the maintenance of cytochrome P450-catalysed activities upon thawing and incubation has been largely described in rat and human PCLS cryopreserved by different methods, whereas several studies demonstrate that phase II enzyme activities (sulfation, glucuronidation…), which are more dependent on the energy status of hepatocytes, are more susceptible to cryopreservation [3,[5][6][7][8]. De Graaf et al [9] observed an improvement of phase II enzyme activities and functionality in PCLS cryopreserved in 18% DMSO as compared to usual concentrations of 10-12% or 30% DMSO.…”

Section: Introductionmentioning

confidence: 99%

“…We have also observed a significant decrease in ATP and glycogen content, and an increase in LDH released in the medium after a few hours of incubation. Others authors report, despite the maintenance of drug metabolism that the absolute values of several viability and functionality parameters are considerably altered in cryopreserved PCLS within 24 hours of incubation [7][8][9][10]. It seemed essential to analyse the biochemical mechanisms underlying the global loss of functionality and integrity of cryopreserved PCLS observed upon incubation after thawing in order to propose scientifically based modifications of culture conditions prone to improve cell viability after thawing.…”

Section: Introductionmentioning

confidence: 99%

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Cryopreservation of Rat Precision-cut Liver Slices is Associated with Major Metabolic Stress and Ionic Perturbations

Vanhulle

Martiat²,

Bontemps³

et al. 2003

Cell Physiol Biochem

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Background/Aims: Cryopreserved precision-cut liver slices (PCLS) in culture exhibit a rapid decline in ATP level and anabolic processes by unknown mechanisms. The aim of this study is to elucidate the key events explaining the alterations occurring in cryopreserved PCLS. Methods: Glucose metabolism, mitochondrial activities, ionic homeostasis and caspase-3 activity were assessed in fresh or cryopreserved (rapid freezing-thawing conditions) rat PCLS within the first hour of incubation in Williams’ medium E at 37°C. Results: Despite a similar glycolytic activity under both conditions, only fresh PCLS were able to gradually recover their ATP and potassium content. Glycogen content dropped more rapidly in cryopreserved than in control PCLS. Only cryopreserved PCLS exhibited a decline in O₂ consumption and a lower ATP/ADP ratio from 30 min of incubation with a loss of coupling of oxygen consumption to ATP synthesis and mitochondrial calcium transport after 1 hour of incubation. Caspase-3 activation was already present in cryopreserved PCLS after a few minutes of incubation. Conclusions: The lack of restoration of potassium/sodium exchange might be a primary event in the metabolic alterations occurring in cryopreserved PCLS after thawing, that contributes to further mitochondrial alterations, caspase activation and compromises ATP-dependent anabolic pathways.

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Section: Discussionmentioning

confidence: 54%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Cryopreservation of Rat Precision-cut Liver Slices is Associated with Major Metabolic Stress and Ionic Perturbations

Vanhulle

Martiat²,

Bontemps³

et al. 2003

Cell Physiol Biochem

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“…In previous studies, we noticed that cryopreserved liver slices with a low viability did not only produce smaller amounts of metabolites, but also the relative amounts of the metabolites changed (unpublished observations). Particularly conjugation activity decreases in unsuccessfully cryopreserved slices, possibly by cofactor loss, whereas phase I biotransformation is preserved longer (Maas et al, 2000;Vanhulle et al, 2001). The selection of slices on the base of their histomorphological appearance after thawing and culturing, as done in the present study, appeared to be useful.…”

Section: Discussionmentioning

confidence: 99%

Comparison of in Vitro Preparations for Semi-Quantitative Prediction of in Vivo Drug Metabolism

Graaf

Meijeren²,

Pektaş³

et al. 2002

Drug Metab Dispos

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ABSTRACT: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 research has been under growing interest for many disciplines within drug safety research but pre-eminently for the selection of animal species as models for human drug toxicity in late drug discovery and early development. In this phase, this is the only way to compare metabolism of a particular drug between animals and humans. From such a species comparison study, the animal species could be selected that forms the same major metabolites as humans do. A prerequisite for making an adequate selection is that the used in vitro system reliably predicts in vivo biotransformation of the drug (i.e., in vivo and in vitro patterns of the major metabolites should be comparable).The in vitro interspecies comparison of drug metabolism approach in early drug development is normally very straightforward to serve speed. In our lab, normally a low and a high drug concentration are used, which are hopefully not too high such that toxicity occurs but high enough to produce detectable amounts of metabolite(s). In this phase, linearity in relation to drug concentration of the metabolite production is not studied, and other possible incubation variables are not optimized, like cell number used and incubation times. Only the major human metabolites are studied for their production by animal preparations.As in vitro tool, both subcellular fractions and intact cell...

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“…Left kidney samples were harvested, washed in 1× phosphate-buffered saline, and snap frozen in ice-cold RPMI-1640 medium (Fisher Scientific, Pittsburgh, Pennsylvania, USA) supplemented with 12% dimethyl sulfoxide. [24] Evaluation of Apoptosis DNA fragmentation analysis was performed as described previously [25] using a fluorescein-FragEL DNA fragmentation detection kit (Oncogene Research Products, Boston, Massachusetts, USA). In brief, paraffin-embedded sections of kidney tissue were deparaffinized and subjected to proteinase K digestion (20 μg/mL).…”

Section: Furosemide I/r Groupmentioning

confidence: 99%

Furosemide Prevents Apoptosis and Associated Gene Expression in a Rat Model of Surgical Ischemic Acute Renal Failure

Aravindan

Riedel

et al. 2007

Renal Failure

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Assessment of Some Critical Factors in the Freezing Technique for the Cryopreservation of Precision-Cut Rat Liver Slices

Cited by 38 publications

References 17 publications

Cryopreservation of Rat Precision-cut Liver Slices is Associated with Major Metabolic Stress and Ionic Perturbations

Cryopreservation of Rat Precision-cut Liver Slices is Associated with Major Metabolic Stress and Ionic Perturbations

Comparison of in Vitro Preparations for Semi-Quantitative Prediction of in Vivo Drug Metabolism

Furosemide Prevents Apoptosis and Associated Gene Expression in a Rat Model of Surgical Ischemic Acute Renal Failure

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