Metabolism of xenobiotics in vitrified human liver slices

544

ABSTRACTS, 26th ANNUAL TABLE l-Abstract

ATP ADP LVP”

26

Group I

GroupII

GroupIII

4.10 f 1.30 1.56 + 0.31 50 + 21

1.06 +- 0.40 1.53 + 0.30 424

4.70 + 1.00 0.99 f 0.18 35 2 25

a Left ventricular pressure.

27. Thermographic Studies of Phantom Kidneys Thawed by Microwave M. K. SCHMEHL, E. F. GRAHAM, KILKOWSKI (Medical University of

and Canine Radiation. AND S. M.

South Carolina, Charleston, South Carolina 29425; and University of Minnesota, St. Paul, Minnesota 55455).

Whole organs, such as kidneys, must be thawed quickly and uniformly to prevent damage during thawing due to excessive heating. Electromagnetic heating with microwaves thaws the kidneys quickly but frequently produces “hot spot” areas with heat damage. To study heat damage, phantom gelatin kidneys with different dielectric constants and canine kidneys perfused with 12.5% glycerol, ethylene glycol, or dimethyl sulfoxide before freezing were microwave thawed and the interior temperature was measured with thermography. The phantom kidneys were thawed freestanding and canine kidneys were either freestanding or packed in a gel mixture. Both phantom and canine kidneys were split symmetrically and separated with a sheet of Styrofoam to facilitate immediate separation and evaluation of the halves after thawing (approximately 3 set). All the phantoms, regardless of dielectric properties, had areas of 37”C after thawing. The freestanding canine kidneys and the gelpacked ethylene glycol-perfused kidneys had frozen areas (37”C). However, glycerol and dimethyl sulfoxide-perfused kidneys packed in gel before thawing had no areas of
Langerhans-A HES and VitrifiKULBE, AND H.

fur Grenzfllchen und Bioverfahrenstechnik, Nobelstrasse 12, 7000 Stuttgart 80, Federal Republic of Germany).

Islets of Langerhans were isolated from pancreata of mice (BALBIC) by digestion with collagenase and DNase at 37°C for 1 hr, filtration through a sieve with a pore width of 500 urn, and sedimentation through Percoll (1.05 g/ml) at lg for I5 min. Before and after

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cryopreservation the islets were cultured for various numbers of days. Viability was tested by insulin release in a perifusion system (2.7 or 22.2 mM glucose in HBS/BSA). The islets were frozen either in RPM1 1640 + 10% FCS or in RPM1 1640 + 10% FCS + 20% HES. After nucleation at - 10°C the samples were plunged into liquid nitrogen (about 3OO”C/min). For vitrification islets were suspended in a solution containing 22.5% dimethyl sulfoxide, 17% acetamide, 11% propylene glycol, 5% polyethylene glycol 6000 (see N.H.P.M. Jutte et al., Cryobiology 24, 294-302, 1987 and plunged directly into liquid nitrogen. In vitrified islets, insulin release was stimulated by high glucose concentration but the increase was less than that of unfrozen islets or islets frozen with HES. 29. Viability and Toxici@ Assessment of Cryopreserved Human Liver Slices. R. FISHER, I. G. SIPES, A. J. GANDOLFI, AND K. BRENDEL

(University of Arizona, Department of Pharmacology and Toxicology, Tucson, Arizona). In order to establish a tissue bank for human in vitro liver toxicological studies, a system was developed for cryopreserving liver slices. Human liver was procured from the Arizona Organ Bank, from the National Disease Research Interchange, or from surgical liver resections. These pieces of liver were placed in a modified Sacks’ solution for transportation to our laboratory. Tissue slices were then generated by using the mechanical Krumdieck tissue slicer. These precisioncut liver slices were cryopreserved in fetal calf serum containing 10% dimethyl sulfoxide by lowering the temperature - I”C/min to -70°C and then directly submerging them into liquid nitrogen. After being stored for 1 day in liquid nitrogen, the slices were thawed by directly submerging them in 37°C fetal calf serum. Fresh and cryopreserved human liver slices were cultured for up to 6 hr and their viability assessed by intracellular K+ retention, protein synthesis, and their ability to respond to ally1 alcohol intoxication. Kf retention and protein synthesis showed that the cryopreserved slices retained between 60-90% of the fresh slice viability. The abilities of fresh and cryopreserved liver slices to respond to increasing concentrations of ally1 alcohol were not significantly different. The intrinsic variation among human tissue specimens, such as genetic parameters, sex, age, concomitant drug therapy and abuse, and the effect of cryopreservation on the multitude of drug metabolic enzymes, requires more extensive comparative studies. However, these preliminary results indicate that human liver slices may be cryopreserved, facilitating their potential use in broad-spectrum human in vitro hepatotoxicity studies. (Supported in part by NIEHS NOl-E5-55112.) 30. Metabolism

of Xenobiotics

in Vitrified

Human

ABSTRACTS,

26th ANNUAL

Liver Slices. S. M. WISHNIES, A. R. PARRISH, I. G. SIPES, A. J. GANDOLFI, AND K. BRENDEL

(Departments of Pharmacology and Toxicology, Health Sciences Center, University of Arizona, Tucson, Arizona). The effects of vitrification, subsequent storage at - l%“C, and thawing (presumably without devitrilication) on the biotransformation of ‘I-hydroxycoumarin (7-HC) and 7-ethoxycoumarin (7-EC) by precision-cut human liver slices in culture were examined. Vitrilication is thought to be the optimal procedure for cryopreservation of biological samples due to the absence of noxious ice-crystal formation. Therefore, our laboratory has pursued vitrification as an alternative to controlled-rate freezing for cryopreserving human liver slices. Liver slices were exposed to increasing concentrations of 1,t-propanediol up to a final concentration of 35% in fetal calf serum. The slices were then blotted and vitrified by direct immersion into liquid N, (LN,) with simultaneous rapid agitation of samples to shake off gas bubbles. Vitrified slices were thawed by percolating a stream of 37°C fetal calf serum over the tissue. Thawing was done either immediately or after 4 and 8 weeks LN, storage. Vitrification and thawing rates were 10,OOOWmin.The ability to biotransform 7-EC to the hydroxy derivative (Phase I cytochrome P450-mediated metabolism) and to the glucuronidation and sulfation (Phase II) of the later substrate was assessed in fresh and vitrified/thawed liver slices. The slices were cultured for up to 6 hr with a nontoxic concentration (50 pm of 7-HC and 7-EC. The culture medium was analyzed for metabolite production and comparisons were made with the biotransformation rate of fresh liver slices at the time of tissue acquisition. Retention of the inherent biotransformation rate was at least 70% when slices were either thawed immediately after vitrification or after 4 and 8 weeks LN, storage. This indicates that storage in LN, did not contribute to the demise of biotransformation activity. In addition phase I and II reactions remained coupled as seen in fresh tissue. Therefore, vitrification may serve as a satisfactory method for cryopreserving human liver slices for investigation of xenobiotic metabolism. (Supported in part by NIEHS NOl-E5-55112.) 31. Cryopreservation

of Hepatocytes: Initial Experience with an Improved Technique to Maintain Long-Term Hepatocyte Function. H. G. KOEBE, J. C. Y. DUNN, M. TONER, A. HUBEL, L. M. STERLING, E. G. CRAVALHO, M. L. YARMUSH, AND R. G. TOMPKINS (Surgical Ser-

vices and Department of Bioengineering, Massachusetts General Hospital, Boston, Massachusetts; and Department of Surgery, Harvard Medical School, Boston, Massachusetts). Current methods for cryopreservation of hepato-

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545

cytes in single-cell suspensions generally result in low overall yields of hepatocytes demonstrating long-term preservation of significant 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. Using this liver cell culture system and standard techniques, adult rat hepatocytes were frozen with 15% Me,SO at a rate of 5Wmin to vapor phase of liquid nitrogen temperature and stored for 30 min. Following rapid thawing, longterm function was assessed by measuring albumin secretion in culture by ELISA methods for 7-14 days postfreezing. Results in the sandwich culture system were compared to cryopreservation of liver cells in single-cell suspensions and in standard monolayer systems. Viable hepatocytes cryopreserved in single-cell suspensions were separated from nonviable cells using Percoll density gradient. Only 2% of the initial liver cells were successfully cultured; they demonstrated albumin secretion rates that were 26-30% of secretion rates for nonfrozen hepatocytes in monolayer culture. This result could not be improved using glycerol, sucrose, or 1,2-propanediol. Freezing liver cells in the sandwich configuration especially after 3 or more days in culture maintained lO-20% of the albumin secretion rates of nonfrozen hepatocytes. Morphology of the cryopreserved liver cells appeared grossly similar to cells without freezing; however, this morphological result was patchy and represented about 3O-l0% of the cells in culture. Of those cells with normal-appearing morphology, minor cytoplasmic membrane damage was evident at the light microscope level but apparently this damage did not affect overall albumin secretion. These results represent the first demonstration of any quantitative long-term preservation of function of hepatocytes by cryopreservation, suggesting that at least a portion of cells survive freezing and maintain their function. 32. Analysis of Cryoprotectant Toxicities for Murine Articular Cartilage. S. WALTSAK, W. A. ELMER, AND K. G. M. BROCKBANK (Biology

Department, Emory University, Atlanta, Georgia) This study was initiated as the first phase in the assessment of chondroitin sulfate synthesis in murine articular cartilage in vitro as an experimental model for the development of prototype clinical cartilage cryopreservation protocols. Cryoprotectant toxicity was assessed by placing neonatal knee joints in either control medium or one of five concentrations of cryoprotectant at 0 and 23 or 37°C. After a 1 hr incubation, the cryoprotectants were removed and the knee joints were washed and labeled with 35S.After 24 hr at 37”C, 35S incorporation into chondroitin sulfate was assessed by scintillation counting and the data expressed