Cold- and cryopreservation of dog liver and kidney slices.

The use of tissue slices in culture could decrease the number of animals used in health-related research and decrease experimental variation. This reduction may come about particularly if the methods of cold- and cryopreserving tissue slices are perfected, and one can conduct sequential in vitro experiments into xenobiotic metabolism, organ-specific toxicity, or organ-specific biochemical processes with tissue slices. With this goal in mind, dog liver and kidney slices were placed in cold storage at 0 degrees C using Viaspan (UW), Euro-Collins (EC), Sacks + prostacyclin (SP), and V-7 (V7) cold-preservation solutions for 10 days. Viability was assessed each day by measuring K+ content and protein synthesis after 4 h of incubation in Waymouth + 10% fetal calf serum (FCS). Dog liver slices can be cold-preserved in V7 for up to 7 days using K+ retention as the viability criterion but only up to 4 days using protein synthesis. Dog kidney slices can be cold-preserved in UW, EC, and V7 for up to 10 days using K+ retention, but only V7 could maintain protein synthesis for 10 days. Cryopreserved dog liver and kidney slices retained 63-68% of control viability after 4 h of incubation in FCS. The cryopreservation regimen included using 10% dimethyl sulfoxide in FCS as the cryoprotectant, a freezing rate of 0.5 degrees C/min for liver slices and 12 degrees C/min for kidney slices, and thawing in 37 degrees C FCS. Continued development of cold- and cryopreserving tissue slices could reduce the numbers of animals used and provide accurate and reproducible data.

Toxicity of cisplatin and mercuric chloride in human kidney cortical slices.

1. Organ specific toxicity such as nephrotoxicity is often investigated with the use of in vivo or in vitro animal models. 2. It would be beneficial if these findings could be verified in a human in vitro system which utilizes non-transplantable human kidneys. 3. Non-transplantable human kidneys were decapsulated, cut in half along the long axis, cores made perpendicular to the hemisphere, and precision-cut renal cortical slices produced. 4. These human kidney slices were incubated for 3, 6, 12, 18 and 24 h, viability assessed using intracellular K+ content, protein synthesis and organic ion transport and the potential nephrotoxicity of cisplatin (0.25, 0.5 and 1.0 mM) and mercuric chloride (10, 50 and 100 microM) on these slices were examined. 5. Control human kidney slices were viable for up to 24 h using all viability parameters while a dose- and time-dependent toxic response was seen using both cisplatin and mercuric chloride. 6. Cisplatin was more nephrotoxic in this human in vitro system than in previously investigated in vitro animal systems whereas mercuric chloride was similar in both systems. 7. These results indicate that human renal cortical slices are useful in predicting and verifying potentially nephrotoxic compounds in man.

Comparative toxicity of valproic acid and its metabolites in liver slices from adult rats, weanling rats and humans.

The incidence of fatal hepatic failure associated with valproic acid (VPA) treatment is low but when it occurs, 90% of the affected patients are below the age of 20 yr. At present the mechanism of VPA hepatotoxicity is unclear; however, it may be a combination of the formation and action of toxic metabolites in developing tissues. In the study reported here we investigated the action of VPA and its metabolites in liver slices prepared from adult and weanling Sprague-Dawley rats and from human livers (non-transplantable livers from organ donors, or biopsy material from patients undergoing surgical liver resection). VPA, 2-propyl-2-pentenoic acid (2-en-VPA or Delta(2)-VPA), 2-propyl-4-pentenoic acid (4-en-VPA or Delta(4)-VPA) were incubated for various times at concentrations of 100 or 300 mug/ml. Protein synthesis and K(|) content were used to assess functional integrity or general viability. The question addressed was whether there were differences in the in vitro toxicity of VPA and its metabolites that were related to the age of the livers from which the slices were taken. Liver slices from weanling rats were significantly more sensitive to VPA and its metabolites than the slices from livers of adult rats. The rank order of toxicities (4-en-VPA > VPA > 2-en-VPA) was the same in both sets of rat slices. The human liver slices were significantly affected by VPA and its metabolites but these compounds were equal in their ability to produce this toxicity. There was also an indication of differences in sensitivity to the toxicity of VPA in slices from livers of donors of different ages.

Cold- and cryopreservation of human liver and kidney slices.

Tissue slices may provide a rapid and economical way of determining cold ischemic effects on human liver and kidney cell viability and metabolism. In contrast to isolated hepatocyte cultures, tissue slices offer an in vitro system which more closely resembles the in vivo situation because of the differentiation and functional heterogeneity of the slice. In this study, human liver and kidney slices were cold stored for 10 days in Belzers University of Wisconsin (UW), Euro-Collins, and Modified Sacks solutions. Another set of slices was cryopreserved at 1 degree C/min for liver and 12 degrees C/min for kidney using a 10% dimethyl sulfoxide/fetal calf serum (FCS) cryoprotectant solution. The cold- and cryopreserved slices were incubated in roller culture for 4 h using FCS as the media. Liver slice viability was assessed by K+ content, protein synthesis, gluconeogenesis, and urea synthesis. Kidney slice viability was assessed using K+ content, protein synthesis, and organic ion transport (PAH and TEA). Human kidney slices were cold preserved in UW for 4-6 days, while the human liver slices were preserved for 12-24 h depending on the viability parameter. Following cryopreservation, human liver slice viability was retained at between 65 and 90% of control values, while kidney slice viability was maintained between 70 and 90% of control values depending on the viability parameter. These results indicate that this human in vitro tissue slice system can be used to optimize preservation solutions and methods. The ability to cold- and cryopreserve human slices could facilitate the more efficient utilization of human tissue.