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Biotransformation activity in vitrified human liver slices
CRYOBIOLOGY
28, 216-226 (19%)
Biotransformation
Activity in Vitrified
Human Liver Slices
S. M. WISHNIES,* A. R. PARRISH,* I. G. SIPES,* A. J. GANDOLFI,* C. W. PUTNAM,* C. L. KRUMDIECK,1AND K. BRENDEL* *Departments of Pharmacology and Toxicology, Health Sciences Center, University of Arizona, Tucson, Arizona M724, and fDepartment of Nutrition Sciences, University of Alabama, Birmingham, Alabama 35294 In vitro testing of human liver for biotransformation or xenobiotic metabolism studies has been limited by unpredictable acquisition of samples. Consequently, it has become necessary to consider methods to cryopreserve and store these samples whenever they do become available for culture of the revived tissue at a more convenient time. Human liver slices were cryopreserved by vitrification, which allows for the transfer of aqueous media to low temperatures (- 196°C) without the formation of ice crystals. Human liver slices were exposed to increasing concentrations of 1,Zpropanediol up to a final concentration of 4.76 M in fetal calf serum. Slices were then vitrified by direct immersion into liquid nitrogen and warmed by submersion in 37°C fetal calf serum. Warming was done either immediately or after 4 and 8 weeks of storage under liquid nitrogen. The effects of vitrification, storage time, and warming on biotransformation were determined by assessing the integrated metabolism of 7ethoxycoumarin (7-EC). Vitrified or fresh human liver slices were exposed to 50 PM 7-EC and its primary metabohte 7-hydroxycoumarin (7-HC) in organ culture for up to 6 hr. Metabolite production of both fresh and vitrified liver slices was compared. Retention of the inherent biotransformation rate was usually high and seemed independent of storage time. Integration of both cytochrome P450-mediated and secondary conjugation processes was retained in cryopreserved tissue. Vitrification offers a way to cryopreserve human liver slices for the study of xenobiotic metabolism in humans. 0 1991 Academic Press, Inc.
In vitro testing of human liver samples may elucidate activities and mechanisms of biotransformation or xenobiotic metabolism that are different or absent in laboratory animals. The use of human liver slices in culture provides a system of high physiological integration which may serve as a valuable tool for predicting metabolic and toxicological responses in humans. However, acquisition of metabolically active human liver tissue from donors is highly unpredictable. The alternative option of using tissue obtained during elective surgical resection often yields metabolically impaired tissue. Therefore, it has become necessary to develop methodologies to cryopreserve viable human tissue. It should be desirable to store large numbers of individual slices from each tissue so that a variety of xenobiotics can be tested upon warming. Most Received February 26, 1990; accepted June 12, 1990.
research in this area has employed the use of suspensions of isolated hepatocytes from both humans and animals as the functional tissue “unit” (15, 16, 19). In contrast, we have developed and used precision-cut human liver slices in combination with organ culture as an alternative in vitro system. As compared to isolated hepatocytes precision-cut liver slices retain the multicellular composition and histological orientation of the parent tissue. This approach might be preferable because it is thought to minimize damage to the tissue and maintain cell-to-cell interactions and avoids the use of potentially deleterious proteolytic enzymes. We have already demonstrated the use of precision-cut animal liver slices in organ-specific toxicity (6, 20-22) and have applied this procedure to human liver tissue (26). Liver slices can rapidly and reproducibly be generated under physiological buffer using a mechanical tissue slicer (11). These slices can then be 216
0011-2240191 $3.00 Copyright 0 1991 by Academic Press, Inc. AU rights of reproduction in any form reserved.
VITRIFIED
HUMAN
cryopreserved in liquid nitrogen. Vitritication was chosen over controlled-rate freezing because of the comparative ease of this procedure in this study. Controlled-rate freezing has the advantage of lower cryoprotectant concentration but is more time consuming and requires more sophisticated cooling equipment. The major difference between the two procedures, of course, is the avoidance of potentially harmful ice crystals in the vitrification process. We report the development of a simple method for vitrifying human liver slices to determine the effects of short-term storage at - 196°C on the retention of integrated xenobiotic metabolism. MATERIALS
AND METHODS
Liver procurement and cold storage. Human liver samples were obtained from the Arizona Organ Bank, the National Disease Research Interchange, or the International Institute for the Advancement of Medicine from cadaveric kidney donors or surgical liver resections. Liver samples were received from the organ banks in ice-cold Euro-Collin’s solution (Euro-Med Inc., Dallas, TX). Resected liver tissue was obtained locally and placed in ice-cold Sack’s (14) solution. Liver slice preparation. Liver samples were cut into 3-cm slabs, cored with a finely sharpened stainless-steel coring tube (l-cm id.), and mechanically sliced in 4°C Krebsbicarbonate, pH 7.4, as described by Smith et al. (23, 24). Two hundred slices (1 cm diameter x 200-250 km thickness) could be generated in approximately 30 min. As soon as a batch of 57 slices became available they were entered into the cryopreservation procedure. Cryopreservation. Fetal calf serum was purchased from Irvine Scientific (Irvine, CA) and 1,Zpropanediol (gold label 99% purity) from Aldrich Chemical Company (Milwaukee, WI). Cryoprotectant exposure/storage trays were specially fabricated to accommodate the human liver slices.
LIVER SLICES
217
The tissue to be vitrified was placed onto a honeycomb-like tray with a capacity of 57 slices. A curved spatula was used to transfer the tissue to the tray (Fig. 1). Both tissue and tray were submerged in 4°C Krebsbicarbonate, pH 7.4. After loading, the trays were lifted out of the Krebs buffer, quickly blotted on absorbent paper to remove excess buffer, then exposed stepwise to increasing concentrations of 1,2-propanediol (1.2, 2.4, and 4.7 M) in fetal calf serum in shallow baths, and kept on ice. The extent of osmotic damage that might occur during cryoprotectant exposure has not been determined. Trays of slices were exposed to each increasing concentration of cryoprotectant for 5 min at 0°C on a gyratory shaker (Fig. 2). Trays with slices were blotted between each transfer to the next higher concentration of cryoprotectant solution to prevent dilution of each solution. After exposure to the last cryoprotectant solution trays were blotted again and rapidly submerged into a large Dewar containing liquid nitrogen under rapid lateral waving to remove N, bubbles that might otherwise insulate the liver slices. In this system slices vitrified in approximately 2 sec. Vitrification was checked by visual inspection as partial vitrification produces noticeable white spots (ice nucleation) on
FIG. 1. Honeycomb exposure and storage tray (nylon mesh fabric laminated to white polystyrene lattice frame) with loading spatula.
218
WISHNIES
ET AL.
Warming. Slices were warmed immediately (i.e., after 2-10 min) or after 4 and 8 weeks of storage by immersion and gentle shaking in a shallow vessel (approx 3 set) containing fetal calf serum maintained at 37°C by a water bath. It is conceivable that during warming a transitory ice film forms on the slice and it is even possible that devitrification takes place, but since all of this happens within approximately 1-2 set we have not been able to analyze the various stages through which the tissue might pass during the rewarming period. We have, however, observed a temporary haze clouding the slices during warming, possibly indicating devitrification or formation FIG. 2. Apparatus for stepwise exposure of liver of a film of ice on the slice. Cracking during slices to increasing concentrations of cryoprotectant. warming has been observed but happens the otherwise translucent slices (Fig. 3). only occasionally. Any changes occurring in osmotic pressure seemed to have been Vitrified slices appeared clear and glasslike and in many regards similar to fresh slices well tolerated by the tissue. After warming, trays with slices were immediately transwet with buffer. ferred into ice-cold fetal calf serum for 15 Storage. Vitrified slices were transferred min to remove the 1,Zpropanediol prior to in their honeycomb tissue carriers to a liqorgan culture. The fetal calf serum was exuid nitrogen storage tank (Cryomed Model LL450) and stored submerged (- 196°C) for changed twice during this period. Removal durations of 2-10 min, 4 weeks, and 8 weeks. of the cryoprotectant was done at low temperature to reduce its toxicity (O’C). Organ culture system. The organ culture system was designed by our laboratory for the incubation of precision-cut tissue slices. Detailed descriptions of the design and application of this system and the tissue slicer have been published elsewhere (11, 22). Briefly, two liver slices were floated onto stainless-steel mesh rollers which were then blotted prior to being placed into scintillation vials containing 1.7 ml of KrebsHepes buffer. The loaded vials were placed on a roller incubator at 37°C and rotated at 1 rpm. Xenobiotics metabolism. 7-EthoxyFIG. 3. Human liver slices maintained on storage coumarin (7-EC) and ‘I-hydroxycoumarin tray just above liquid N,. (a) slice exposed to 1.2 M (7-HC) were purchased from Aldrich (pu1,Zpropanediol for 5 mitt (frozen). (b) Slice exposed as rity 99%). Stock solutions of 7-EC and 7in (a), then to 2.4 M for another 5 min (partially vitris&oxide fied). (c) Slice exposed as in (b), then to 4.7 M for HC were prepared in dimethyl (Me,SO). Each chemical was then diluted another 5 min (vitrified). Light spots visible on slice c are not ice nucleation centers but reflections of light. in Krebs-Hepes (a Krebs-bicarbonate so-
VITRIFIED
HUMAN
lution in which the bicarbonate has been replaced by N-2-hydroxyethylpiperazine N*-Zethanesulfonic acid), pH 7.45, to a final concentration of 50 $l4 (0.1% Me,SO was found to be without any effects in our organ culture system). Before exposure to 7-EC or 7-HC fresh, vitrified liver slices were preincubated in Krebs-Hepes buffer at 37°C for 1 hr. After preincubation the slices were transferred into fresh vials (2 slices/vial) containing 1.7 ml of either 50 p,iW 7-HC or 50 @W 7-EC in Krebs-Hepes. They were then returned to organ culture for up to 4 or 6 hr at 37°C. Liver slice protein was determined by a method modified after Lowry (12). Aliquots of the medium (100 ~1) were removed from each vial at 2, 4, and 6 hr and stored at -80°C until analyzed by HPLC for the presence of 7-HC and 7-EC metabolites (Fig. 4). Analysis of 7-HC and 7-EC metabolites. Tetrahydrofuran and isopropyl alcohol were purchased from EM Science (Cherry Hill, NJ). A Spectra Physics HPLC system (San Jose, CA) which consists of a SP 8800 pump, SP 8775 autosampler, SP 8450 UV
219
LIVER SLICES
detector, and an SP 4290 integrator, was used for all analyses. Sample aliquots were centrifuged at 11,000 rpm for 5 min and then directly analyzed by HPLC. The assay procedure, as developed by our laboratory, employs a 5-brn, 25 x 0.46-cm internal surface reversed phase (ISRP) column (Regis Chemical Co.) to isocratically resolve 7EC, 7-HC, and the glucuronide and sulfate of 7-HC. The mobile phase used was 4% isopropyl alcohol, 1% tetrahydrofuran, and 95% KH*PO, 0.1 M buffer (pH 6.0) at a flow rate of 1 ml per minute. The UV detector was set at the optimal wavelength for both parent and metabolites (320 nm). Both conjugates of 7-HC were identified by enzyme hydrolysis using specific p-glucuronidase and sulfatase and 35S incorporation into the sulfate peak. Sulfate and glucuronide conjugates of 7-HC were expressed in terms of 7-HC equivalents since standards were not available. RESULTS
Both 7-EC and 7-HC were used to assess the metabolic integrity of fresh and vitrified
‘I-Ethoxycoumrrin (Phase
I)
Cytochrome
P450
+
7-Hydroxycoumarin Glucuronidation
1
1
.,......,,.,m0
Sulfatlon
sulfateAmo (Phase
ill
FIG. 4. 7-Ethoxycoumarin
metabolism.
220
WISHNIES
human liver slices. The integrated metabolism of 7-EC was used as a sensitive indicator of cytochrome P450-mediated metabolism (phase I) and its coupling to secondary conjugation reactions (phase II). 7-HC was used to directly assess phase II metabolism (glucuronidation and sulfation). Because of the marked variability seen in the metabolism of 7-EC and 7-HC between human livers data are presented separately for each liver sample. At this point all observations on minor differences in qualitative aspects are disregarded and only the major metabolites were considered. The kinetic data of 7-EC and 7-HC metabolism from a typical cryopreservation experiment are shown in Figs. 5 and 6. It can be seen that both fresh and vitrified human liver slices form 7-HC glucuronide as the major metabolite when incubated with either substrate (7-EC or 7-HC). In both cases very little of
ET AL.
the sulfate conjugate is observed but the small rate of formation seemed not to be affected by cryopreservation or storage. When 7-EC was used as the substrate there appeared to be a slight increase in the amount of 7-HC metabolite produced by vitrified slices in comparison to fresh slices. This increase was observed in spite of a quasi-constant production of the conjugate. Tables l-3 reflect the fact that both fresh and vitrified liver slices usually have similar kinetics when 7-EC or 7-HC is metabolized. This appears to hold true for vitrified liver slices even after 4 and 8 weeks of LN2 storage (Tables 3 and 4). More importantly, vitrified slices maintain the coupling between phases I and II of metabolism of 7EC. Interestingly, sulfate production is very low and nonlinear compared to glucuronidation in both fresh and cryopreserved tissue. This observation could be 15 lb
0
2
4
6
0
2
A/
4
Incubation
lime
(hrs)
lncubotion
Incubation
Time
(hrs)
Incubation
6
Time
(hn)
lime
(hm)
FIG. 5. Typical kinetics of 7-EC metabolism in liver slices from human donor C: fresh (not vitrified) (a); vittied (2-10 min) (b); vitrified and stored for 4 (c) and 8 (d) weeks under LN,. Fresh and warmed slices were maintained in organ culture for 6 hr. Each data point reflects the average metabolites produced in duplicate culture vials (two slices/vial). l ,7-HC-glucuronide; A, 7-HC; 0, 7-HC-sulfate.
VITRIFIED
Cl0
2
4 lncubotion
Time
lime
Incubation
HUMAN
221
LIVER SLICES
0
6 (hm)
(hrs)
2
6
4 Incubation
lime
(hm)
Incubation
lime
(hn)
FIG. 6. Typical kinetics of 7-HC metabolism in liver slices from human donor C: fresh (not vitrilied) (a); vitrified (2-10 min) (b); vitrified and stored for 4 (c) and 8 (d) weeks under LN,. Fresh and warmed slices were maintained in organ culture for 6 hr. Each data point reflects the average metabolites produced in duplicate culture vials (two slices/vial). l , 7-HC-glucuronide; 0, 7-HC-sulfate.
due to a limitation imposed by the buffer composition (i.e., low sulfate content). In contrast production of 7-HC-glucuronide is nearly linear over time. The most important result is that both major metabolic steps, i.e., 0-deethylation
of 7-EC and the glucuronidation of the intermediary 7-HC, seemed not to be diminished in vitrified slices that were stored for up to 8 weeks. Vitrification as compared to freezing (i.e., an amorphous glass in contrast to a
TABLE 1 Human Donor A Incubation time (hd Fresh slices Vitrified slices, LNZ storage 2-10 min
Metabolite production (nmol/mg protein) 7-HC-glucuronide
‘I-HC-sulfate
7-HC
2 4
l.% 6.51
1.12 2.25
0.32 1.07
2 4
1.03 3.37
0.65 1.67
0.75 1.07
Note. Fresh and vitrified human liver slices were exposed to 50 uM7-EC for up to 4 hr in organ culture at 37°C. Metabolite production in medium was analyzed by HPLC. Each data point reflects the average metabolites produced in duplicate culture vials (two slices/vial).
222
WISHNIES
ET AL.
TABLE 2 Human Resection A
Fresh slices
Incubation time b-1 2 4
Vitrified slices, LN2 storage 2-10 min
Metabolite production (nmol/mg protein) %HC-ghrcuronide 0.49 1.34
2 4
0.42 1.24
7-H&sulfate 0.64 0.85 0.71 0.71
7-HC 0 0.27 0.32 0.83
Nore. Fresh and vitrified human liver slices were exposed to either 50 @4 7-HC or 7-EC for up to 4 hr in organ culture at 37°C. Metabolite production in medium was analyzed by HPLC. Each data point reflects the average metabolites produced in duplicate culture vials (two slices/vial). LINot determined.
multicrystalline structure) in slices is demonstrated in Fig. 3, which shows frozen and vitrified slices side by side on their carrier tray and photographed at - 150°C. This comparison shows that it is easily possible to distinguish frozen (white opaque) from vitrified (brown glasslike) slices simply by visual inspection. DISCUSSION
Cryopreservation of biological systems by vitrification is a recent and rapidly growing development in cryobiology. Vitrilication requires high and potentially toxic concentrations of cryoprotectants. In our procedure slices in cryoprotectant solutions were cooled in a nonequilibrium mode so that they would solidify into an amorphous glass state (13). It is possible that fetal calf serum helps in achieving this state under conditions which otherwise do not lead to vitrification. The effect of fetal calf serum might be due to inhibition of ice nucleation. Several aqueous solutions for vitrification have been described (4, 5, 13) and some have been successfully applied to cryopreserve mouse embryos (18), rat blastocysts (lo), human and rat islets of Langerhans (8, 17), and human monocytes (25). This procedure is thought to be advantageous since the damaging effects of intra- and extracellular ice crystallization are avoided. Also, vitrification is a relatively simple and con-
venient procedure for the cryopreservation of small tissue samples (9). Optimal conditions for vitrification demand manipulating a number of variables: (1) cryoprotectant type and concentration; (2) vehicle medium; (3) time, temperature, and stepping of the exposure to the cryoprotectant; (4) cooling rate; (5) storage time; (6) warming rate; and (7) stepwise removal of cryoprotectant . Successful cryopreservation should yield tissue samples that retain a useful level of metabolic activity comparable to that of fresh liver for biotransformation studies. Hawkins et al. showed that small tissue aggregates such as rabbit kidney slices could be cryopreserved with dimethyl sulfoxide, glycerol, and ethylene glycol in such a way that viability was partially retained (7). In three other studies utilizing controlled freezing procedures, cooling rates, cryoprotectant concentrations, and media were altered to reduce damage to rabbit kidney slices from cryoprotectant toxicity and intra- and extracellular ice-crystal formation (l-3). However, the intrinsic damaging effects of ice crystals on cell membranes could not be completely avoided. In this paper we describe a procedure for vitrifying human liver slices with the marker of viability being integrated metabolism of a model substrate, 7-EC. The major metabolite of 7-EC, 7-HC, was also
VITRIFIED
HUMAN
LIVER
223
SLICES
TABLE 3 Human Donor B Incubation time
W
Metabolite production (nmol/mg protein) 7-HC-glucuronide
7-HC-sulfate
7-HC
50 pM 7-EC Fresh slices Vitrified slices, LN, storage 2-10 min 4 weeks 8 weeks
50pM 7-HC
Fresh slices
Vitrified slices, LN, storage 2-10 min 4 weeks 8 weeks
2 4 6
3.52 8.73 11.96
0 0.20 0.41
0.91 1.69 1.83
2 4 6 2(n = 1) 4 6(n = 1) 2 4 6
1.47 1.93 2.43 25.66 0 31.42 29.14 34.41 43.85
0 0 0 0 0 0 0 0 0
1.69 3.38 3.90 0.4 D 0.4 1.27 1.59 1.59
2 4 6
15.73 35.09 61.61
0.48 1.20 1.20
-
1.32 0.80 1.32 26.06 28.06 31.27 19.43 28.12 31.21
0 0 0 0 0 0 1.50 2.31 2.99
-
2 4 (n 6 2(n 4(n 6 (n 2 4 6
= 1) = 1) = 1) = 1)
Note. Fresh and vitrified human liver slices were exposed to either 50 @f 7-HC or 7-EC for up to 6 hr in organ culture at 37°C. Metabolite production in medium was analyzed by HPLC. Each data point reflects the average metabolites produced in duplicate culture vials (two slices/vial). a Not determined.
monitored. The high concentrations of cryoprotectant required seemed not to grossly alter the kinetics of the metabolism of these chemicals. Preliminary experiments were done in which slices were exposed to various concentrations of 1 ,Zpropanediol at room temperature and at 0°C. We found that liver slices could tolerate exposure to higher concentrations of 1,2-propanediol at 0°C than at room temperature as measured by potassium retention as an indicator of slice viability (data not shown). To reduce
the extent of damage during exposure to the cryoprotectant others have introduced the cryoprotectant in steps of increasing concentration while simultaneously lowering exposure temperature prior to vitrification (3,8, 18). We used a stepwise approach but kept the temperature during cryoprotectant exposure to a constant 0°C to minimize the toxic effects of the 1,Zpropanediol. Upon warming we did not use a stepwise dilution to protect against osmotic damage but did not see any deleterious effects. Mixtures of
224
WISHNIES
TABLE
ET AL.
4
Human Donor C Metabolite production Incubation time (hr) 50 @%I7-EC Fresh slices Vitrified slices, LN, storage 2-10 min 4 weeks 8 weeks 50 pM 7-HC Fresh slices Vitrified slices LN, storage 2-10 min 4 weeks 8 weeks
7-HC-glucuronide nmohmg protein
‘I-HC-sulfate nmol/mg protein
7-HC mnol/mg protein
2 4 6
4.80 -+ 0.28 8.64 f 0.42 11.95 f 1.64
0.65 + 0.46 1.30 k 0.24 1.30 + 0.24
0.26 + 0.26 0.69 f 0.35 0.69 f 0.35
2 4 6 2 (n = 1) 4 6(n = 1) 2 4 6
3.36 6.34 8.22 2.29 6.57 14.60 3.56 7.22 12.56
0.33 0.67 0.67 0.20 0.58 0.58
0.67 1.33 1.44 1.28 2.72 5.01 1.10 2.46 3.63
2 4 6
20.45 + 3.16 31.90 f 4.86 37.66 2 5.51
1.00 f 0.16 1.12 f 0.28 0.95 f 0.11
-
13.42 18.46 21.56 16.05 27.08 33.22 11.52 18.81 27.00
0.43 0.64 0.32 1.12 1.45 1.45 0.92 1.18 1.03
-
2 4(n 6 2(n 4(n 6(n 2 4 6
= 1) = 1) = 1) = 1)
+ 0.12 2 0.62 + 1.54 2 0.75 f f 5.52 f 0.53 f 0.88 -t- 3.97
f 3.16 f 4.47 f 6.24 f 2.52 f 1.91 f 3.24 2 0.46 +- 2.08 2 l.%
f f f f 2 + a 0 0.39 2
0.33 0.01 0.01 0.20 0.58 0.58 0.39
f 0.01 * 0.20 + 0.10 * 0.45 ” 0.12 -+ 0.12 k 0.41 f 0.15 f -
f f ” + 2 2 + f +
0.23 0.45 0.34 0.51 0.39 0.57 0.06 0.14 0.53
Note. Fresh and vitrified human liver slices were exposed to either 50 pM 7-HC or 7-EC for up to 6 h in organ culture at 37°C. Metabolite production in medium was analyzed by HPLC. Each data point reflects the average metabolites produced in duplicate culture vials + SE. a Not determined.
cryoprotectants containing additives such as acetamide have been used by others to counter the toxic effects of high concentrations of dimethyl sulfoxide (18) [for a review of vitrification solutions see (4,5, 13)]. We have used a different approach to counter toxicity by using the relatively nontoxic 1,Zpropanediol instead of Me,SO. The very low concentration of Me,SO (0.1%) used in the drug exposure incubations was without any measurable effect.
Fetal calf serum as a vehicle solution gave the best results in parallel experiments where slices were cryopreserved by controlled freezing and was therefore also used in this study. We have vitrified preconditioned and blotted slices by rapid (i.e., >5000°C/min) lateral immersion into liquid nitrogen. Upon warming these slices partially retained xenobiotic metabolism. Both cytochrome Pdsr,-mediated O-deethylation (phase I) and glucuronidation and sulfation
VITRIFIED
HUMAN
reactions (phase II) remained coupled, with the 7-HC-glucuronide being the major metabolite formed. We have shown that the metabolic profiles and the level of metabolic activity for fresh and vitrified liver slices are very similar. Research with dog hepatocytes using a method of controlledrate freezing indicated that the sulfation of hydroxybiphenyl may be a sensitive indicator of cryoinjury and was found to be decreased in cryopreserved tissue (15). In tissues examined by us very little sulfate conjugate was produced in fresh slices but comparable amounts were seen upon cryopreservation. In several cases we observed higher metabolic rates in the vitrified tissue as compared to fresh tissue. This phenomenon remains without explanation at this point but selective increases in metabolic activity in cryopreserved human liver have been reported by others (16). Phase II conjugation reactions would be expected to incur major damage due to inadequate cryopreservation because damaged cell membranes would allow cofactors for these reactions to leak out. We therefore feel that the presence of coupling of integrated metabolism serves as an important indicator of functional viability. Phase I metabolism might be more resistant to cofactor leakage than phase II. With the scarcity of human tissue for research the development of optimal cryopreservation procedures is extremely important. Successful cryopreservation should allow the establishment of tissue banks to investigate xenobiotic metabolism and toxicity in human tissue in vitro where in vivo research is impossible or unethical.
LIVER
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ACKNOWLEDGMENTS
We gratefully acknowledge the support of this work by NIEHS Contract NIH NOl-ES-55112 and the helpful discussions with the project officer, Dr. L. T. Burka at NIEHS. We also thank the Arizona Organ Bank, Mr. T. Hagan in Phoenix and Mr. L. Burnett in Tucson, the National Disease Research Interchange, and the International Institute for the Advancement of Medicine for human tissue made available to us. Ad-
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WISHNIES
13. McFarlane, D. R. Physical aspects of vitrification in aqueous solutions. Cryobiology 24, 181-195 (1987). 14. Monden, M., and Fortner, J. G. Twenty-four and forty-eight hour canine liver preservation by simple hypothermia with prostacycline. Ann. Surg. l%, 3&12 (1982). 15. Powis, G., Santone, K. S., Melder, D. C., Thomas, L., Moore, D. J., and Wilke, T. J. Cryopreservation of rat and dog hepatocytes for studies of xenobiotic metabolism and activation. Drug Metab. Dispos. 15, 826-832 (1987). 16. Powis, G., Jardine, I., Van Dyke, R., Weinshilbaum, R., Moore, D., Wilke, T., Rhodes, W., Nelson, R., Benson, L., and Szumlanski, C. Foreign compound metabolism studies with human liver obtained as surgical waste: Relation to donor characteristics and effects of tissue storage. Drug Metab. Dispos. 16, 582-589 (1988). 17. Rajotte, R. V., De Groot, T. J., Ellis, D. K., and Rall, W. F. Preliminary experiments on vitrilication of isolated rat islets of Langerhans. Cryobiol. Abstr. 22, 602 (1985). 18. Rall, W. F., and Fahy, G. M. Ice-free cryopreservation of mouse embryos at - 196°C by vitrilication. Nature (London) 313, 573-575 (1985). 19. Runtjes, P. J., Moshage, H. J., Van Gemert, P. J., De Waal, R., and Yap, S. H. Cryopreservation of adult human hepatocytes. The inlluence of deep freezing storage on the viability, cell seeding survival, line structures and albumin synthesis in primary cultures. J. Hepatol. 3, 7-18 (1986).
ET AL. 20. Sipes, I. G., Fisher, R. L., Smith, P. F., Stine, E. R., Gandolli, A. J., and Brendel, K. A dynamic liver culture system: A tool for studying chemical biotransformation and toxicity. Me&. Models Toxicol. Arch. Toxicol. Suppl. 11, 2033 (1987). 21. Smith, P. F., Gandolli, A. J., Krumdieck, C. L., and Brendel, K. A novel organ culture system for the maintenance of adult rat liver. Fed. Proc. 44, 627 (1985). 22 Smith, P. F., Shubat, P. J., Gandolfi, A. J., ’ Krumdieck, C. L., and Brendel, K. Novel dynamic organ culture system for hepatotoxicity studies. Toxicologist 5, 151 (1985). 23 Smith, P. F., Krack, G., McKee, R. L., Johnson, ’ D. G., Gandolfi, A. J., Hruby, V. J., Krumdieck, C. L., and Brendel, K. Maintenance of adult rat liver slices in dynamic organ culture. In Vitro Cell. Dev. 32, 706-712 (1986). 24. Smith, P. F., Gandolfi, A. J., Krumdieck, C. L., Putnam, C. W., Zukowski, C. F., Davis, W. M., and Brendel, K. Organ culture of precision-cut liver slices for in vitro toxicology. Life Sci. 36, 1367-1375 (1985). Takahashi, T., Hirsh, A., Erbe, E. F., Bross, 25. J. B., Steere, R. L., and Williams, R. J. Vitrification of human monocytes. Cryobiology 23, 103-115 (1986). 26. Wishnies, S. M., Parrish, A. R., Sipes, I. G., Gandolli, A. J., and Brendel, K. Partial retention of biotransformation in cryopreserved precision cut liver slices. Toxicologist 8, 203 (1988).
28, 216-226 (19%)
Biotransformation
Activity in Vitrified
Human Liver Slices
S. M. WISHNIES,* A. R. PARRISH,* I. G. SIPES,* A. J. GANDOLFI,* C. W. PUTNAM,* C. L. KRUMDIECK,1AND K. BRENDEL* *Departments of Pharmacology and Toxicology, Health Sciences Center, University of Arizona, Tucson, Arizona M724, and fDepartment of Nutrition Sciences, University of Alabama, Birmingham, Alabama 35294 In vitro testing of human liver for biotransformation or xenobiotic metabolism studies has been limited by unpredictable acquisition of samples. Consequently, it has become necessary to consider methods to cryopreserve and store these samples whenever they do become available for culture of the revived tissue at a more convenient time. Human liver slices were cryopreserved by vitrification, which allows for the transfer of aqueous media to low temperatures (- 196°C) without the formation of ice crystals. Human liver slices were exposed to increasing concentrations of 1,Zpropanediol up to a final concentration of 4.76 M in fetal calf serum. Slices were then vitrified by direct immersion into liquid nitrogen and warmed by submersion in 37°C fetal calf serum. Warming was done either immediately or after 4 and 8 weeks of storage under liquid nitrogen. The effects of vitrification, storage time, and warming on biotransformation were determined by assessing the integrated metabolism of 7ethoxycoumarin (7-EC). Vitrified or fresh human liver slices were exposed to 50 PM 7-EC and its primary metabohte 7-hydroxycoumarin (7-HC) in organ culture for up to 6 hr. Metabolite production of both fresh and vitrified liver slices was compared. Retention of the inherent biotransformation rate was usually high and seemed independent of storage time. Integration of both cytochrome P450-mediated and secondary conjugation processes was retained in cryopreserved tissue. Vitrification offers a way to cryopreserve human liver slices for the study of xenobiotic metabolism in humans. 0 1991 Academic Press, Inc.
In vitro testing of human liver samples may elucidate activities and mechanisms of biotransformation or xenobiotic metabolism that are different or absent in laboratory animals. The use of human liver slices in culture provides a system of high physiological integration which may serve as a valuable tool for predicting metabolic and toxicological responses in humans. However, acquisition of metabolically active human liver tissue from donors is highly unpredictable. The alternative option of using tissue obtained during elective surgical resection often yields metabolically impaired tissue. Therefore, it has become necessary to develop methodologies to cryopreserve viable human tissue. It should be desirable to store large numbers of individual slices from each tissue so that a variety of xenobiotics can be tested upon warming. Most Received February 26, 1990; accepted June 12, 1990.
research in this area has employed the use of suspensions of isolated hepatocytes from both humans and animals as the functional tissue “unit” (15, 16, 19). In contrast, we have developed and used precision-cut human liver slices in combination with organ culture as an alternative in vitro system. As compared to isolated hepatocytes precision-cut liver slices retain the multicellular composition and histological orientation of the parent tissue. This approach might be preferable because it is thought to minimize damage to the tissue and maintain cell-to-cell interactions and avoids the use of potentially deleterious proteolytic enzymes. We have already demonstrated the use of precision-cut animal liver slices in organ-specific toxicity (6, 20-22) and have applied this procedure to human liver tissue (26). Liver slices can rapidly and reproducibly be generated under physiological buffer using a mechanical tissue slicer (11). These slices can then be 216
0011-2240191 $3.00 Copyright 0 1991 by Academic Press, Inc. AU rights of reproduction in any form reserved.
VITRIFIED
HUMAN
cryopreserved in liquid nitrogen. Vitritication was chosen over controlled-rate freezing because of the comparative ease of this procedure in this study. Controlled-rate freezing has the advantage of lower cryoprotectant concentration but is more time consuming and requires more sophisticated cooling equipment. The major difference between the two procedures, of course, is the avoidance of potentially harmful ice crystals in the vitrification process. We report the development of a simple method for vitrifying human liver slices to determine the effects of short-term storage at - 196°C on the retention of integrated xenobiotic metabolism. MATERIALS
AND METHODS
Liver procurement and cold storage. Human liver samples were obtained from the Arizona Organ Bank, the National Disease Research Interchange, or the International Institute for the Advancement of Medicine from cadaveric kidney donors or surgical liver resections. Liver samples were received from the organ banks in ice-cold Euro-Collin’s solution (Euro-Med Inc., Dallas, TX). Resected liver tissue was obtained locally and placed in ice-cold Sack’s (14) solution. Liver slice preparation. Liver samples were cut into 3-cm slabs, cored with a finely sharpened stainless-steel coring tube (l-cm id.), and mechanically sliced in 4°C Krebsbicarbonate, pH 7.4, as described by Smith et al. (23, 24). Two hundred slices (1 cm diameter x 200-250 km thickness) could be generated in approximately 30 min. As soon as a batch of 57 slices became available they were entered into the cryopreservation procedure. Cryopreservation. Fetal calf serum was purchased from Irvine Scientific (Irvine, CA) and 1,Zpropanediol (gold label 99% purity) from Aldrich Chemical Company (Milwaukee, WI). Cryoprotectant exposure/storage trays were specially fabricated to accommodate the human liver slices.
LIVER SLICES
217
The tissue to be vitrified was placed onto a honeycomb-like tray with a capacity of 57 slices. A curved spatula was used to transfer the tissue to the tray (Fig. 1). Both tissue and tray were submerged in 4°C Krebsbicarbonate, pH 7.4. After loading, the trays were lifted out of the Krebs buffer, quickly blotted on absorbent paper to remove excess buffer, then exposed stepwise to increasing concentrations of 1,2-propanediol (1.2, 2.4, and 4.7 M) in fetal calf serum in shallow baths, and kept on ice. The extent of osmotic damage that might occur during cryoprotectant exposure has not been determined. Trays of slices were exposed to each increasing concentration of cryoprotectant for 5 min at 0°C on a gyratory shaker (Fig. 2). Trays with slices were blotted between each transfer to the next higher concentration of cryoprotectant solution to prevent dilution of each solution. After exposure to the last cryoprotectant solution trays were blotted again and rapidly submerged into a large Dewar containing liquid nitrogen under rapid lateral waving to remove N, bubbles that might otherwise insulate the liver slices. In this system slices vitrified in approximately 2 sec. Vitrification was checked by visual inspection as partial vitrification produces noticeable white spots (ice nucleation) on
FIG. 1. Honeycomb exposure and storage tray (nylon mesh fabric laminated to white polystyrene lattice frame) with loading spatula.
218
WISHNIES
ET AL.
Warming. Slices were warmed immediately (i.e., after 2-10 min) or after 4 and 8 weeks of storage by immersion and gentle shaking in a shallow vessel (approx 3 set) containing fetal calf serum maintained at 37°C by a water bath. It is conceivable that during warming a transitory ice film forms on the slice and it is even possible that devitrification takes place, but since all of this happens within approximately 1-2 set we have not been able to analyze the various stages through which the tissue might pass during the rewarming period. We have, however, observed a temporary haze clouding the slices during warming, possibly indicating devitrification or formation FIG. 2. Apparatus for stepwise exposure of liver of a film of ice on the slice. Cracking during slices to increasing concentrations of cryoprotectant. warming has been observed but happens the otherwise translucent slices (Fig. 3). only occasionally. Any changes occurring in osmotic pressure seemed to have been Vitrified slices appeared clear and glasslike and in many regards similar to fresh slices well tolerated by the tissue. After warming, trays with slices were immediately transwet with buffer. ferred into ice-cold fetal calf serum for 15 Storage. Vitrified slices were transferred min to remove the 1,Zpropanediol prior to in their honeycomb tissue carriers to a liqorgan culture. The fetal calf serum was exuid nitrogen storage tank (Cryomed Model LL450) and stored submerged (- 196°C) for changed twice during this period. Removal durations of 2-10 min, 4 weeks, and 8 weeks. of the cryoprotectant was done at low temperature to reduce its toxicity (O’C). Organ culture system. The organ culture system was designed by our laboratory for the incubation of precision-cut tissue slices. Detailed descriptions of the design and application of this system and the tissue slicer have been published elsewhere (11, 22). Briefly, two liver slices were floated onto stainless-steel mesh rollers which were then blotted prior to being placed into scintillation vials containing 1.7 ml of KrebsHepes buffer. The loaded vials were placed on a roller incubator at 37°C and rotated at 1 rpm. Xenobiotics metabolism. 7-EthoxyFIG. 3. Human liver slices maintained on storage coumarin (7-EC) and ‘I-hydroxycoumarin tray just above liquid N,. (a) slice exposed to 1.2 M (7-HC) were purchased from Aldrich (pu1,Zpropanediol for 5 mitt (frozen). (b) Slice exposed as rity 99%). Stock solutions of 7-EC and 7in (a), then to 2.4 M for another 5 min (partially vitris&oxide fied). (c) Slice exposed as in (b), then to 4.7 M for HC were prepared in dimethyl (Me,SO). Each chemical was then diluted another 5 min (vitrified). Light spots visible on slice c are not ice nucleation centers but reflections of light. in Krebs-Hepes (a Krebs-bicarbonate so-
VITRIFIED
HUMAN
lution in which the bicarbonate has been replaced by N-2-hydroxyethylpiperazine N*-Zethanesulfonic acid), pH 7.45, to a final concentration of 50 $l4 (0.1% Me,SO was found to be without any effects in our organ culture system). Before exposure to 7-EC or 7-HC fresh, vitrified liver slices were preincubated in Krebs-Hepes buffer at 37°C for 1 hr. After preincubation the slices were transferred into fresh vials (2 slices/vial) containing 1.7 ml of either 50 p,iW 7-HC or 50 @W 7-EC in Krebs-Hepes. They were then returned to organ culture for up to 4 or 6 hr at 37°C. Liver slice protein was determined by a method modified after Lowry (12). Aliquots of the medium (100 ~1) were removed from each vial at 2, 4, and 6 hr and stored at -80°C until analyzed by HPLC for the presence of 7-HC and 7-EC metabolites (Fig. 4). Analysis of 7-HC and 7-EC metabolites. Tetrahydrofuran and isopropyl alcohol were purchased from EM Science (Cherry Hill, NJ). A Spectra Physics HPLC system (San Jose, CA) which consists of a SP 8800 pump, SP 8775 autosampler, SP 8450 UV
219
LIVER SLICES
detector, and an SP 4290 integrator, was used for all analyses. Sample aliquots were centrifuged at 11,000 rpm for 5 min and then directly analyzed by HPLC. The assay procedure, as developed by our laboratory, employs a 5-brn, 25 x 0.46-cm internal surface reversed phase (ISRP) column (Regis Chemical Co.) to isocratically resolve 7EC, 7-HC, and the glucuronide and sulfate of 7-HC. The mobile phase used was 4% isopropyl alcohol, 1% tetrahydrofuran, and 95% KH*PO, 0.1 M buffer (pH 6.0) at a flow rate of 1 ml per minute. The UV detector was set at the optimal wavelength for both parent and metabolites (320 nm). Both conjugates of 7-HC were identified by enzyme hydrolysis using specific p-glucuronidase and sulfatase and 35S incorporation into the sulfate peak. Sulfate and glucuronide conjugates of 7-HC were expressed in terms of 7-HC equivalents since standards were not available. RESULTS
Both 7-EC and 7-HC were used to assess the metabolic integrity of fresh and vitrified
‘I-Ethoxycoumrrin (Phase
I)
Cytochrome
P450
+
7-Hydroxycoumarin Glucuronidation
1
1
.,......,,.,m0
Sulfatlon
sulfateAmo (Phase
ill
FIG. 4. 7-Ethoxycoumarin
metabolism.
220
WISHNIES
human liver slices. The integrated metabolism of 7-EC was used as a sensitive indicator of cytochrome P450-mediated metabolism (phase I) and its coupling to secondary conjugation reactions (phase II). 7-HC was used to directly assess phase II metabolism (glucuronidation and sulfation). Because of the marked variability seen in the metabolism of 7-EC and 7-HC between human livers data are presented separately for each liver sample. At this point all observations on minor differences in qualitative aspects are disregarded and only the major metabolites were considered. The kinetic data of 7-EC and 7-HC metabolism from a typical cryopreservation experiment are shown in Figs. 5 and 6. It can be seen that both fresh and vitrified human liver slices form 7-HC glucuronide as the major metabolite when incubated with either substrate (7-EC or 7-HC). In both cases very little of
ET AL.
the sulfate conjugate is observed but the small rate of formation seemed not to be affected by cryopreservation or storage. When 7-EC was used as the substrate there appeared to be a slight increase in the amount of 7-HC metabolite produced by vitrified slices in comparison to fresh slices. This increase was observed in spite of a quasi-constant production of the conjugate. Tables l-3 reflect the fact that both fresh and vitrified liver slices usually have similar kinetics when 7-EC or 7-HC is metabolized. This appears to hold true for vitrified liver slices even after 4 and 8 weeks of LN2 storage (Tables 3 and 4). More importantly, vitrified slices maintain the coupling between phases I and II of metabolism of 7EC. Interestingly, sulfate production is very low and nonlinear compared to glucuronidation in both fresh and cryopreserved tissue. This observation could be 15 lb
0
2
4
6
0
2
A/
4
Incubation
lime
(hrs)
lncubotion
Incubation
Time
(hrs)
Incubation
6
Time
(hn)
lime
(hm)
FIG. 5. Typical kinetics of 7-EC metabolism in liver slices from human donor C: fresh (not vitrified) (a); vittied (2-10 min) (b); vitrified and stored for 4 (c) and 8 (d) weeks under LN,. Fresh and warmed slices were maintained in organ culture for 6 hr. Each data point reflects the average metabolites produced in duplicate culture vials (two slices/vial). l ,7-HC-glucuronide; A, 7-HC; 0, 7-HC-sulfate.
VITRIFIED
Cl0
2
4 lncubotion
Time
lime
Incubation
HUMAN
221
LIVER SLICES
0
6 (hm)
(hrs)
2
6
4 Incubation
lime
(hm)
Incubation
lime
(hn)
FIG. 6. Typical kinetics of 7-HC metabolism in liver slices from human donor C: fresh (not vitrilied) (a); vitrified (2-10 min) (b); vitrified and stored for 4 (c) and 8 (d) weeks under LN,. Fresh and warmed slices were maintained in organ culture for 6 hr. Each data point reflects the average metabolites produced in duplicate culture vials (two slices/vial). l , 7-HC-glucuronide; 0, 7-HC-sulfate.
due to a limitation imposed by the buffer composition (i.e., low sulfate content). In contrast production of 7-HC-glucuronide is nearly linear over time. The most important result is that both major metabolic steps, i.e., 0-deethylation
of 7-EC and the glucuronidation of the intermediary 7-HC, seemed not to be diminished in vitrified slices that were stored for up to 8 weeks. Vitrification as compared to freezing (i.e., an amorphous glass in contrast to a
TABLE 1 Human Donor A Incubation time (hd Fresh slices Vitrified slices, LNZ storage 2-10 min
Metabolite production (nmol/mg protein) 7-HC-glucuronide
‘I-HC-sulfate
7-HC
2 4
l.% 6.51
1.12 2.25
0.32 1.07
2 4
1.03 3.37
0.65 1.67
0.75 1.07
Note. Fresh and vitrified human liver slices were exposed to 50 uM7-EC for up to 4 hr in organ culture at 37°C. Metabolite production in medium was analyzed by HPLC. Each data point reflects the average metabolites produced in duplicate culture vials (two slices/vial).
222
WISHNIES
ET AL.
TABLE 2 Human Resection A
Fresh slices
Incubation time b-1 2 4
Vitrified slices, LN2 storage 2-10 min
Metabolite production (nmol/mg protein) %HC-ghrcuronide 0.49 1.34
2 4
0.42 1.24
7-H&sulfate 0.64 0.85 0.71 0.71
7-HC 0 0.27 0.32 0.83
Nore. Fresh and vitrified human liver slices were exposed to either 50 @4 7-HC or 7-EC for up to 4 hr in organ culture at 37°C. Metabolite production in medium was analyzed by HPLC. Each data point reflects the average metabolites produced in duplicate culture vials (two slices/vial). LINot determined.
multicrystalline structure) in slices is demonstrated in Fig. 3, which shows frozen and vitrified slices side by side on their carrier tray and photographed at - 150°C. This comparison shows that it is easily possible to distinguish frozen (white opaque) from vitrified (brown glasslike) slices simply by visual inspection. DISCUSSION
Cryopreservation of biological systems by vitrification is a recent and rapidly growing development in cryobiology. Vitrilication requires high and potentially toxic concentrations of cryoprotectants. In our procedure slices in cryoprotectant solutions were cooled in a nonequilibrium mode so that they would solidify into an amorphous glass state (13). It is possible that fetal calf serum helps in achieving this state under conditions which otherwise do not lead to vitrification. The effect of fetal calf serum might be due to inhibition of ice nucleation. Several aqueous solutions for vitrification have been described (4, 5, 13) and some have been successfully applied to cryopreserve mouse embryos (18), rat blastocysts (lo), human and rat islets of Langerhans (8, 17), and human monocytes (25). This procedure is thought to be advantageous since the damaging effects of intra- and extracellular ice crystallization are avoided. Also, vitrification is a relatively simple and con-
venient procedure for the cryopreservation of small tissue samples (9). Optimal conditions for vitrification demand manipulating a number of variables: (1) cryoprotectant type and concentration; (2) vehicle medium; (3) time, temperature, and stepping of the exposure to the cryoprotectant; (4) cooling rate; (5) storage time; (6) warming rate; and (7) stepwise removal of cryoprotectant . Successful cryopreservation should yield tissue samples that retain a useful level of metabolic activity comparable to that of fresh liver for biotransformation studies. Hawkins et al. showed that small tissue aggregates such as rabbit kidney slices could be cryopreserved with dimethyl sulfoxide, glycerol, and ethylene glycol in such a way that viability was partially retained (7). In three other studies utilizing controlled freezing procedures, cooling rates, cryoprotectant concentrations, and media were altered to reduce damage to rabbit kidney slices from cryoprotectant toxicity and intra- and extracellular ice-crystal formation (l-3). However, the intrinsic damaging effects of ice crystals on cell membranes could not be completely avoided. In this paper we describe a procedure for vitrifying human liver slices with the marker of viability being integrated metabolism of a model substrate, 7-EC. The major metabolite of 7-EC, 7-HC, was also
VITRIFIED
HUMAN
LIVER
223
SLICES
TABLE 3 Human Donor B Incubation time
W
Metabolite production (nmol/mg protein) 7-HC-glucuronide
7-HC-sulfate
7-HC
50 pM 7-EC Fresh slices Vitrified slices, LN, storage 2-10 min 4 weeks 8 weeks
50pM 7-HC
Fresh slices
Vitrified slices, LN, storage 2-10 min 4 weeks 8 weeks
2 4 6
3.52 8.73 11.96
0 0.20 0.41
0.91 1.69 1.83
2 4 6 2(n = 1) 4 6(n = 1) 2 4 6
1.47 1.93 2.43 25.66 0 31.42 29.14 34.41 43.85
0 0 0 0 0 0 0 0 0
1.69 3.38 3.90 0.4 D 0.4 1.27 1.59 1.59
2 4 6
15.73 35.09 61.61
0.48 1.20 1.20
-
1.32 0.80 1.32 26.06 28.06 31.27 19.43 28.12 31.21
0 0 0 0 0 0 1.50 2.31 2.99
-
2 4 (n 6 2(n 4(n 6 (n 2 4 6
= 1) = 1) = 1) = 1)
Note. Fresh and vitrified human liver slices were exposed to either 50 @f 7-HC or 7-EC for up to 6 hr in organ culture at 37°C. Metabolite production in medium was analyzed by HPLC. Each data point reflects the average metabolites produced in duplicate culture vials (two slices/vial). a Not determined.
monitored. The high concentrations of cryoprotectant required seemed not to grossly alter the kinetics of the metabolism of these chemicals. Preliminary experiments were done in which slices were exposed to various concentrations of 1 ,Zpropanediol at room temperature and at 0°C. We found that liver slices could tolerate exposure to higher concentrations of 1,2-propanediol at 0°C than at room temperature as measured by potassium retention as an indicator of slice viability (data not shown). To reduce
the extent of damage during exposure to the cryoprotectant others have introduced the cryoprotectant in steps of increasing concentration while simultaneously lowering exposure temperature prior to vitrification (3,8, 18). We used a stepwise approach but kept the temperature during cryoprotectant exposure to a constant 0°C to minimize the toxic effects of the 1,Zpropanediol. Upon warming we did not use a stepwise dilution to protect against osmotic damage but did not see any deleterious effects. Mixtures of
224
WISHNIES
TABLE
ET AL.
4
Human Donor C Metabolite production Incubation time (hr) 50 @%I7-EC Fresh slices Vitrified slices, LN, storage 2-10 min 4 weeks 8 weeks 50 pM 7-HC Fresh slices Vitrified slices LN, storage 2-10 min 4 weeks 8 weeks
7-HC-glucuronide nmohmg protein
‘I-HC-sulfate nmol/mg protein
7-HC mnol/mg protein
2 4 6
4.80 -+ 0.28 8.64 f 0.42 11.95 f 1.64
0.65 + 0.46 1.30 k 0.24 1.30 + 0.24
0.26 + 0.26 0.69 f 0.35 0.69 f 0.35
2 4 6 2 (n = 1) 4 6(n = 1) 2 4 6
3.36 6.34 8.22 2.29 6.57 14.60 3.56 7.22 12.56
0.33 0.67 0.67 0.20 0.58 0.58
0.67 1.33 1.44 1.28 2.72 5.01 1.10 2.46 3.63
2 4 6
20.45 + 3.16 31.90 f 4.86 37.66 2 5.51
1.00 f 0.16 1.12 f 0.28 0.95 f 0.11
-
13.42 18.46 21.56 16.05 27.08 33.22 11.52 18.81 27.00
0.43 0.64 0.32 1.12 1.45 1.45 0.92 1.18 1.03
-
2 4(n 6 2(n 4(n 6(n 2 4 6
= 1) = 1) = 1) = 1)
+ 0.12 2 0.62 + 1.54 2 0.75 f f 5.52 f 0.53 f 0.88 -t- 3.97
f 3.16 f 4.47 f 6.24 f 2.52 f 1.91 f 3.24 2 0.46 +- 2.08 2 l.%
f f f f 2 + a 0 0.39 2
0.33 0.01 0.01 0.20 0.58 0.58 0.39
f 0.01 * 0.20 + 0.10 * 0.45 ” 0.12 -+ 0.12 k 0.41 f 0.15 f -
f f ” + 2 2 + f +
0.23 0.45 0.34 0.51 0.39 0.57 0.06 0.14 0.53
Note. Fresh and vitrified human liver slices were exposed to either 50 pM 7-HC or 7-EC for up to 6 h in organ culture at 37°C. Metabolite production in medium was analyzed by HPLC. Each data point reflects the average metabolites produced in duplicate culture vials + SE. a Not determined.
cryoprotectants containing additives such as acetamide have been used by others to counter the toxic effects of high concentrations of dimethyl sulfoxide (18) [for a review of vitrification solutions see (4,5, 13)]. We have used a different approach to counter toxicity by using the relatively nontoxic 1,Zpropanediol instead of Me,SO. The very low concentration of Me,SO (0.1%) used in the drug exposure incubations was without any measurable effect.
Fetal calf serum as a vehicle solution gave the best results in parallel experiments where slices were cryopreserved by controlled freezing and was therefore also used in this study. We have vitrified preconditioned and blotted slices by rapid (i.e., >5000°C/min) lateral immersion into liquid nitrogen. Upon warming these slices partially retained xenobiotic metabolism. Both cytochrome Pdsr,-mediated O-deethylation (phase I) and glucuronidation and sulfation
VITRIFIED
HUMAN
reactions (phase II) remained coupled, with the 7-HC-glucuronide being the major metabolite formed. We have shown that the metabolic profiles and the level of metabolic activity for fresh and vitrified liver slices are very similar. Research with dog hepatocytes using a method of controlledrate freezing indicated that the sulfation of hydroxybiphenyl may be a sensitive indicator of cryoinjury and was found to be decreased in cryopreserved tissue (15). In tissues examined by us very little sulfate conjugate was produced in fresh slices but comparable amounts were seen upon cryopreservation. In several cases we observed higher metabolic rates in the vitrified tissue as compared to fresh tissue. This phenomenon remains without explanation at this point but selective increases in metabolic activity in cryopreserved human liver have been reported by others (16). Phase II conjugation reactions would be expected to incur major damage due to inadequate cryopreservation because damaged cell membranes would allow cofactors for these reactions to leak out. We therefore feel that the presence of coupling of integrated metabolism serves as an important indicator of functional viability. Phase I metabolism might be more resistant to cofactor leakage than phase II. With the scarcity of human tissue for research the development of optimal cryopreservation procedures is extremely important. Successful cryopreservation should allow the establishment of tissue banks to investigate xenobiotic metabolism and toxicity in human tissue in vitro where in vivo research is impossible or unethical.
LIVER
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ACKNOWLEDGMENTS
We gratefully acknowledge the support of this work by NIEHS Contract NIH NOl-ES-55112 and the helpful discussions with the project officer, Dr. L. T. Burka at NIEHS. We also thank the Arizona Organ Bank, Mr. T. Hagan in Phoenix and Mr. L. Burnett in Tucson, the National Disease Research Interchange, and the International Institute for the Advancement of Medicine for human tissue made available to us. Ad-
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