Morphological and biochemical integrity of human liver slices in long-term culture: effects of oxygen tension.

We tested the effects of low (20% O2) and high (70% O2) oxygen tension on the morphological and biochemical integrity of human liver slices incubated for up to 72 h in supplemented Williams' E medium in a dynamic rotating culture system. High oxygen tension was more effective than low oxygen tension for preserving morphological integrity in long-term culture (48-72 h). After 72 h of culture with 70% O2, the lobular pattern was well preserved, and the survival of hepatocytes (approximately 80%) and other cell types was good. Immunohistochemical studies showed good preservation of the region-specific expression of CYP2EI and CYP3A4 isoenzymes for up to 72 h of incubation in 70% O2. As compared to 20% O2, the oxidized glutathione content and reactive oxygen species production were slightly increased in 70% O2, suggesting that minimal oxidative stress occurred with the high oxygen tension. In conclusion, despite slight oxidative stress associated with high oxygen tension, 70% O2 appeared more appropriate than 20% O2 for preserving the morphological and biochemical integrity of human liver slices cultured in a dynamic organ culture system for up to 72 h.

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.

Cyclin A2 and c-myc mRNA expression in ethinyl estradiol induced liver proliferation.

The time-course of c-myc and cyclin A2 mRNA expression was determined in the liver of male Sprague-Dawley rats during transient liver cell proliferation induced by a single dose of ethinyl estradiol (EE), and was compared to that during liver regeneration following two-thirds hepatectomy (PH). Cell proliferation was assessed in terms of 5'-bromodeoxyuridine (BrdU) labeling. EE administration and PH both increased BrdU labeling between 18 and 48 h, with peak values at 18 and 24 h. An early (2 h) increase in BrdU labeling was observed after EE but not PH. Maximal increases in cyclin A2 mRNA levels and BrdU labeling coincided after both EE and PH, and cyclin A2 mRNA expression was proportional to the intensity of the proliferative response. In contrast, the degree of c-myc mRNA expression was similar after EE administration and PH, but the time course was different: c-myc gene expression rose concomitantly with DNA replication after EE, while after PH it increased during the prereplicative phase. This indicates that the pattern of c-myc gene expression in the liver is strongly related to the type of proliferative response.

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 degrees 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.

Precision-cut dog renal cortical slices in dynamic organ culture for the study of cisplatin nephrotoxicity.

The dog is the non-rodent species the most often used in preclinical drug safety evaluation. In this study, we established a new system of precision-cut dog renal cortical slices, evaluated their biochemical, functional, and morphological integrity, and determined the effects of cisplatin (cis-diamminedichloroplatinum (II), CDDP), a very potent nephrotoxic antineoplastic agent used to treat a variety of solid tumors, on the viability and histopathology of slices. Precision-cut renal cortical slices were made perpendicular to the cortical-papillary axis. Slices were incubated in DMEM/Ham's F12 culture medium containing 1 g/L glucose, 2 mmol/L glutamine, and 2 mmol/L heptanoic acid at 37 degrees C in an atmosphere of 5% CO2-70% O2-25% N2 in dynamic organ culture. Our results showed that slices maintained ATP and GSH content, protein synthesis, Na(+)-dependent uptake of glucose inhibited by phlorizin, PAH (p-aminohippuric acid) uptake inhibited by probenecid, and TEA (tetraethylammonium) uptake inhibited by mepiperphenidol for at least 6 h of culture, and morphological integrity up to 24 h. After 6 h of exposure, CDDP induced vacuolation and cell necrosis in the epithelial tubular cells of slices with a concentration-related increase in extension but not in severity. The development of the lesions started in the proximal tubules and extended to the distal tubules. The location and the extension of the lesions confirmed the observations in dog kidneys after in vivo treatment with CDDP by the intravenous route. The concentration-related decrease in slice viability after 6 h exposure to CDDP was in keeping with the extension of the histopathological lesions in the renal parenchyma. The slice viability was unaffected up to 0.63 mmol/L CDDP. At 1.25 and 2.5 mmol/L CDDP, slice viability fell by 35% and 75%, respectively. These results suggest that precision-cut dog renal cortical slices in culture may be suitable for addressing the specific nephrotoxicity issues encountered in this species.