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%.     

Assessment of some critical factors in the freezing technique for the cryopreservation of precision-cut rat liver slices

A number of studies on the cryopreservation of precision-cut liver slices using various techniques have been reported. However, the identification of important factors that determine cell viability following cryopreservation is difficult because of large differences between the various methods published. The aim of this study was to evaluate some important factors in the freezing process in an effort to find an optimized approach to the cryopreservation of precision-cut liver slices. A comparative study of a slow and a fast freezing technique was carried out to establish any differences in tissue viability for a number of endpoints. Both freezing techniques aim at the prevention of intracellular ice formation, which is thought to be the main cause of cell death after cryopreservation. Subsequently, critical variables in the freezing process were studied more closely in order to explain the differences in viability found in the two methods in the first study. For this purpose, a full factorial experimental design was used with 16 experimental groups, allowing a number of variables to be studied at different levels in one single experiment. It is demonstrated that ATP and K(+) content and histomorphology are sensitive parameters for evaluating slice viability after cryopreservation. Subsequently, it is shown that freezing rate and the cryopreservation medium largely determine the residual viability of liver slices after cryopreservation and subsequent culturing. It is concluded that a cryopreservation protocol with a fast freezing step and using William's Medium E as cryopreservation medium was the most promising approach to successful freezing of rat liver slices of those tested in this study.     

Effects of cryoprotectant addition and washout methods on the viability of precision-cut liver slices

Successful vitrification of organ slices is hampered by both osmotic stress and chemical toxicity of cryoprotective agents (CPAs). In the present study, we focused on the effect of osmotic stress on the viability of precision-cut liver slices (PCLS) by comparing different CPA solutions and different methods of loading and unloading the slices with the CPAs. For this purpose, we developed a gradient method to load and unload CPAs with the intention of minimizing sudden changes in osmolarity and thereby avoiding osmotic stress in the slices in comparison with the commonly used step-wise loading/unloading approach. With this gradient method, the CPA solution was introduced at a constant rate into a specially designed mixing chamber containing the slices. We showed that immediate mixing of the infused CPA and the chamber constituents occurred, which enabled us to control the CPA concentration to which PCLS were exposed as a function of time.With this method, CPA concentration versus time profiles were varied using various commercially available CPA mixtures [VMP, VM3, M22, and modified M22 (mM22)]. The viability of PCLS was determined after CPA loading and unloading and subsequent incubation during 3 h at 37 °C. Despite the reduction of osmotic stress, the viability of slices did not improve with gradual loading and unloading and remained considerably lower than that of untreated slices. The toxicity of the three CPA solutions did not correlate with either their potential osmotic effects or their total concentrations, and did not change strongly with exposure time in 100% CPA. The most likely explanation for these observations is that PCLS are not very sensitive to osmotic changes of the magnitude imposed in our study, and chemical toxicity of the CPA solutions is the main barrier to be overcome. The chemical toxicity of the CPAs used in this study probably originates from a source other than the total concentration of the solutions. The presented gradient method using the specially designed chamber is more time and cost effective than the step-wise method and can be universally applied to efficiently evaluate different CPA solutions.     

Effects of cryoprotectant addition and washout methods on the viability of precision-cut liver slices

Successful vitrification of organ slices is hampered by both osmotic stress and chemical toxicity of cryoprotective agents (CPAs). In the present study, we focused on the effect of osmotic stress on the viability of precision-cut liver slices (PCLS) by comparing different CPA solutions and different methods of loading and unloading the slices with the CPAs. For this purpose, we developed a gradient method to load and unload CPAs with the intention of minimizing sudden changes in osmolarity and thereby avoiding osmotic stress in the slices in comparison with the commonly used step-wise loading/unloading approach. With this gradient method, the CPA solution was introduced at a constant rate into a specially designed mixing chamber containing the slices. We showed that immediate mixing of the infused CPA and the chamber constituents occurred, which enabled us to control the CPA concentration to which PCLS were exposed as a function of time.     

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°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°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°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.     

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.     

Effects of CYP inhibitors on precocene I metabolism and toxicity in rat liver slices

We present a comprehensive in vitro approach to assessing metabolism-mediated hepatotoxicity using male Sprague–Dawley rat liver slices incubated with the well characterized hepatotoxicant, precocene I, and inhibitors of cytochrome P450 (CYP) enzymes. This approach combines liquid chromatography mass spectrometry (LC MS) detection methods with multiple toxicity endpoints to enable identification of critical metabolic pathways for hepatotoxicity. The incubations were performed in the absence and presence of the non-specific CYP inhibitor, 1-aminobenzotriazole (ABT) and isoform-specific inhibitors. The metabolite profile of precocene I in rat liver slices shares some features of the in vivo profile, but also had a major difference in that epoxide dihydrodiol hydrolysis products were not observed to a measurable extent. As examples of our liver slice metabolite identification procedure, a minor glutathione adduct and previously unreported 7-O-desmethyl and glucuronidated metabolites of precocene I are reported. Precocene I induced hepatocellular necrosis in a dose- and time-dependent manner. ABT decreased the toxicity of precocene I, increased exposure to parent compound, and decreased metabolite levels in a dose-dependent manner. Of the isoform-specific CYP inhibitors tested for an effect on the precocene I metabolite profile, only tranylcypromine was noticeably effective, indicating a role of CYPs 2A6, 2C9, 2Cl9, and 2E1. With respect to toxicity, the order of CYP inhibitor effectiveness was ABT > diethyldithiocarbamate∼tranylcypromine > ketoconazole. Furafylline and sulfaphenazole had no effect, while quinidine appeared to augment precocene I toxicity. These results suggest that rat liver slices do not reproduce the reported in vivo biotransformation of precocene I and therefore may not be an appropriate model for precocene I metabolism. However, these results provide an example of how small molecule manipulation of CYP activity in an in vitro model can be used to confirm metabolism-mediated toxicity.     

Modulation of paracetamol metabolism by Kupffer cells: a study on rat liver slices

Recent studies support the hypothesis that non parenchymal cells (mainly macrophages) may play a role in the metabolism and cellular effects of paracetamol. In order to investigate this hypothesis, male Wistar rats were intravenously injected with either 7.5 mg/kg gadolinium chloride (Gd+) or NaCl 0.9% (Gd-). The treatment with GdCl3 decreased the number and the function of Kupffer cells in liver tissue, as assessed by the histological examination of the liver after colloidal carbon injection in the portal vein. Precision-cut liver slices (PCLS) were prepared from both groups of rats and cultured for 8h in Waymouth's medium in the presence and absence of 5 mM paracetamol. Interestingly, PCLS obtained from Gd+ rats exhibited a lower release of tumor necrosis factor (TNF-alpha) and a better viability than PCLS from control (Gd-) rats. Incubation with paracetamol led to a decreased glycogen level in liver slices from Gd+ or Gd-, without modifying neither liver morphology nor ATP level nor LDH release. A higher proportion of paracetamol glucuronide, was secreted from the slices obtained from Gd+ rats. These data suggest that Kupffer cells could affect the viability of PCLS in culture and are involved in the regulation of phase II metabolism in the adjacent hepatocytes. We propose that PCLS in culture is a suitable model to elucidate the biochemical mechanism underlying the modulation of metabolism occurring through hepatocytes-Kupffer cells interactions.     

Role of apoptotic signaling pathway in metabolic disturbances occurring in liver tissue after cryopreservation: Study on rat precision-cut liver slices

Precision-cut liver slices in culture (PCLS) appears as a useful and widely used model for metabolic studies; the interest to develop an adequate cryopreservation procedure, which would allow maintaining cell integrity upon incubation, is needed to extend its use for human tissues. We have previously shown that cryopreservation of rat PCLS leads to caspase-3 activation and early alterations of their K+ content upon incubation. In this study, we tested the hypothesis that counteracting intracellular K+ loss and/or interfering with cell death signaling pathways could improve the viability of cryopreserved PCLS. PCLS were prepared from male Wistar rat liver and cryopreserved by rapid freezing before incubation. The addition of a caspase inhibitor-Z-DEVD-FMK (2.5 microM)-in the culture medium did not improve viability of cryopreserved PCLS. Incubation of cryopreserved PCLS in a K+ rich medium (135 mM) increased K+ content and avoided caspase-3 activation, but did not improve cell viability. Caspase-3 inhibition, a decrease in cell lysis, and improvement of glycogen content were observed in cryopreserved PCLS after addition of LiCl (100 mM) in the incubation medium. These results indicate that, even if caspase-3 activation is linked to the K+ loss in cryopreserved PCLS, its inhibition does not allow restoring the metabolic capacities. LiCl, acting on a target upstream of caspase-3 inhibition, improves cell viability and allows glycogen accumulation when added in culture medium of cryopreserved PCLS; and could thus be considered as an interesting adjuvant in the culture of cryopreserved PCLS.     

Cryopreserved rat liver slices: a critical evaluation of cell viability, histological integrity, and drug-metabolizing enzymes

The effects of a cryopreservation procedure on the biochemical, morphological and functional integrity of rat liver slices just after thawing and after 24 h culture were evaluated. Freshly prepared slices were incubated in modified University of Wisconsin solution containing 50% fetal calf serum and 10% dimethyl sulfoxide for 20 min on ice prior to a rapid cooling in liquid nitrogen. After 10-40 days, slices were thawed rapidly at 42 degrees C. Total protein content and (3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) (MTT) reduction were well preserved at thawing, whereas ATP content was markedly decreased relative to freshly prepared slices (-83%). The major microscopic findings in sections of just-thawed liver slices consisted of hepatocellular dissociation and minimal apoptosis. The qualitative profile of antipyrine (AP) metabolism was well preserved in cryopreserved slices, but the amounts of phase I and phase II AP metabolites produced over a 3-h incubation period were markedly reduced relative to fresh slices (-58 to -71%). When cryopreserved slices were cultured for 24 h after thawing, the viability was markedly reduced, as reflected by the almost complete absence of MTT reduction and the loss of ATP content. Histological examinations showed extensive cellular necrosis. The amount of AP metabolites produced by cryopreserved slices was similar after a 3- or a 24-h culture period, indicating that AP metabolism capacities were lost at 24 h culture. In conclusion, our results suggest that cryopreserved rat liver slices may be a useful model for short-term in vitro determination of drug metabolism pathways. Further work is required to extend their use for toxicological studies.     

Morphological and functional integrity of precision-cut rat liver slices in rotating organ culture and multiwell plate culture: Effects of oxygen tension

We examined the maintenance of functional and morphological integrity of precision-cut rat liver slices cultured in various incubation systems and conditions for 72 h. Slices were incubated (37°C) for 6, 24, 48, and 72 h in supplemented Williams E medium in 6-well plastic culture plates on a gyratory shaking platform (WPCS) or in a rotating organ culture system (ROCS) using 5% CO₂–95% air (WPCS/air or ROCS/air) or 5% CO₂–70% O₂–25% N₂ (WPCS/ O₂ or ROCS/ O₂). Biochemical and functional parameters of slices maintained in WPCS/air or WPCS/ O₂ were almost totally inhibited after 24 h, in keeping with the extensive and diffuse coalescing coagulative necrosis typical of post-ischemic injury affecting almost all the slice surface after 48 h. As compared to freshly isolated slices, slices maintained in ROCS/air for 72 h showed stable ATP and GSH content, increased protein synthesis, and a slight steady decrease in GST activity, while ATP and GST activity remained stable and protein synthesis and GSH content increased in slices incubated in ROCS/ O₂ for 72 h. The extent of coagulative necrosis was markedly lower in longitudinal sections from slices incubated for 72 h in ROCS/ O₂ than in ROCS/air. Transversal sections from slices kept in ROCS/air for 72 h showed a thick central band of necrotic cells edged by two peripheral layers of viable hepatocytes, whereas most of the slice was composed of viable hepatocytes lined by two thin layers of necrotic cells after 72 h in ROCS/ O₂. ROCS/ O₂ emerged as the system best preserving the histological and functional integrity of rat liver slices in long-term culture.     

Effect of excessive intake of ascorbic acid on hepatic and extra-hepatic phase I and phase II drug metabolism in rat

Guinea pig is the animal model of choice for studies on effects of ascorbic acid (AA). However, rat is one of the largely used animals for investigations related to chemical carcinogenesis. Therefore, the present study was designed to evaluate the changes induced by high intake of the vitamin in xenobiotic and carcinogen metabolizing status of the organs. Male Wistar rats, dosed daily with 50 mg AA/100 g body weight for 10 weeks, demonstrated a small non-significant increase in hepatic, pulmonary and colon cytochrome P-450 (Cyt. P-450) contents, which was accompanied with a significant increase in hepatic and pulmonary arylhydrocarbon hydroxylase (AHH) activities. Phase II enzymes of drug metabolism responded in different ways to increased intake of AA. UDP-glucuronyltransferase (UDPGT) activity was unaffected in liver and colon, but it was increased (p less than 0.005) in lung. Activities of glutathione S-transferase (GST) were decreased in the three organs. Inducibility of AHH by 3-methylcholanthrene (MCA) or phenobarbital (PB) was largely reduced due to AA feeding. Besides this, MCA and PB had differential effects on enzymatic levels in AA fed rats. When compared with our earlier observations in guinea pig, it was found that rat responded similarly to guinea pig to increased intake of AA with regard to hepatic AHH, Cyt. P-450, UDPGT and GST, pulmonary AHH, Cyt. P-450 and Cyt. b5, and all studied colon enzymes, except GST.