CRYOBIOLOGY
27, 288-300
(l%‘tl)
Drug Metabolism
and Viability Studies Rat Hepatocytes
in Cryopreserved
J. A. COUNDOURIS,**t M. H. GRANT,* J. G. SIMPSON,S AND G. M. HAWKSWORTH*?t Departments of *Medicine and Therapeutics (Unit qf Clinical Pharmacology), fPharmaco/ogy, and #Pathology, University of Aberdeen, Polwarth Building, Foresterhill, Aberdeen AB9 220, United Kingdom Rat hepatocytes were cryopreserved optimalty by freezing them at 1Wmin to - 80°C in cryoprotectant medium containing either 20% (v/v) diiethylsulfoxide (Me,SO) and 25% (v/v) fetal calf serum in Leibowitz LlS medium (Me,!30 cryoprotectant) or 25% (v/v) vitrification solution (containing Me,SO, acetamide, propylene glycol and polyethylene glycol) in Leibowitz Ll5 medium (VS25). The VS25 solution was superior for maintaining viability during short-term storage (2W hr) but was slightly toxic during longer storage periods (7 days). Although thawed cells were 40-K% viable on ice after cryopreservation, their viability fell rapidly during incubation in suspension at 37°C. This decline in viability occurred more rapidly after freezing in Me,SO cryoprotectant than in VS25 and was associated with extensive intracellular damage and cell swelling. The loss in viability at 37°C does not appear to be due to ice-crystal damage as it occurred in cells stored at - 10°C{above the freezing point of the cryoprotectants) and it may be due to temperature/osmotic shock. Both cryoprotectant media were equally efficient at preserving enzyme activities in the hepatocytes over 7 days at -80°C. Cytochrome P450 and reduced glutathione content and the activities of the microsomal enzymes responsible for aminopyrine N-demethylation and epoxide hydrolysis were well maintained over 7 days storage. In contrast, the cytosolic enzymes glutathione-S-transferase and glutathione reductase were markedly labile during cryopreservation. Cytosolic enzymes may be more susceptible to icecrystal damage, whereas the microsomal membrane may protect the enzymes which are embedded in it.
0 1990 Academic
Press, Inc.
The metabolism and cytotoxic potential of xenobiotics in humans are usually estimated by extrapolation from animal studies. However, there is evidence that marked species differences in drug metabolism exist (27). It is therefore essential to develop systems that can be used to measure the metabolism and predict the toxicity of xenobiotics in humans. Suspensions of human liver cells would provide a defined system for this work as they contain the enzymes responsible for xenobiotic metabolism and simultaneously provide a target cell for the assessment of toxicity. Viable human adult hepatocytes can be isolated by perfusion of liver segments obtained from kidney donor patients (26); liver obtained postmortem is unsuitable for this purpose. However, the supply Received June 9, 1988; accepted July 27, 1989. 288 0011-2240190 Copyright All rights
$3.00
0 IS90 by Academic Pre66, Inc. of reproduction in any form reserved.
of human liver is infrequent and unpredictable. When suitable material is obtained, large numbers (600 x 10”) of hepatocytes are available in suspension but they remain viable for only about 4 hr. Therefore, one of the most urgent practical requirements is the ability to prolong the useful lifetime of these hepatocyte preparations. The approach most frequently used to prolong the lifetime of hepatocytes is primary culture. A major problem inherent in the culture of mammalian hepatocytes is the instability of a number of specific drugmetabolizing enzymes (11). For example, cytochrome P450-dependent mixed function oxidase (MFO) activity declines to low levels within the first 2&8 hr of culture (6, 13). Although the drug-metabolizing enzyme activities of human hepatocytes are more stable during culture than those of rodent hepatocytes, the ratios of MFO activ-
CRYOPRESERVATION
289
OF RAT HEPATOCYTES
ities and conjugation reactions are continuously changing (9). We have, therefore, sought an alternative method of prolonging the useful lifetime of hepatocytes by cryopreservation of the cells. This process has been used previously for the preservation of human oocytes, sperm, embryos, and other cell types. Previous attempts to cryopreserve hepatocytes have shown that dimethylsulfoxide (Me,SO) is the most suitable cryoprotectant available (8, 16, 20). Many xenobiotics are activated to reactive metabolites by the MFO system and detoxified by various conjugation reactions including conjugation with reduced glutathioine (GSH) and glucuronic acid. The extent of cytotoxicity depends on the balance between these activation and detoxification processes. For cryopreserved hepatocytes to act as a valid system for toxicity studies, they must, after thawing, maintain all the activation and detoxification reactions of drug metabolism at levels found in freshly isolated hepatocytes. In this paper we have compared the ability of two Me,SO-based cryoprotectant media to preserve the viability and drugmetabolizing activity of isolated rat hepatocytes. To assess the preservation of the MFO system, the cellular cytochrome P450 content and the N-demethylation of aminopyrine were measured in the hepatocytes postcryopreservation. Intracellular glutathione participates in such diverse biological processes as detoxification of xenobiotits, removal of hydroperoxides, maintenance of sulfhydryl status of proteins, and modulation of enzyme activities by sulfhydry1 exchange. In cells, glutathione is effectively maintained in the reduced state by glutathione reductase, which is linked to the NADPH/NADP+ system, and intracellular GSH homeostasis exerts a major influence on the status of the NADPH pool (23). Measurement of intracellular GSH levels, GSH-S-transferase, and glutathione reductase activities in postcryopreserved hepatocytes gives an indication of their ability to
protect the cells from xenobiotic-induced cytotoxicity. Many cytotoxic and carcinogenic compounds form reactive epoxides (14, 17) which are subsequently detoxified by epoxide hydrolase. We have measured the activity of the microsomal epoxide hydrolase as a further indication of the metabolic competence of the cryopreserved hepatocytes. These studies will subsequently be extended to human hepatocytes once the optimum cryopreservation conditions have been developed. MATERIALS
Isocitric acid, isocitrate dehydrogenase, bovine serum albumin (fraction V), polyethylene glycol (relative molecular mass 8000), Me,SO, NADPH, and reduced glutathione were all obtained from Sigma Chemical Company (Poole, Dorset). NADP and collagenase were from BoehringerMannheim and acetamide and propylene glycol from BDH Ltd. Fetal calf serum was obtained from Gibco and the Leibowitz L15 medium from Flow Laboratories. Phenanthrene-9, lo-oxide and phenanthrene-9, IOdihydrodiol were prepared as previously described (3). METHODS
Preparation, Freezing, of Hepatocytes
and Thawing
Adult rat hepatocytes (viability > 85%, assessed by trypan blue exclusion) were prepared from male Sprague-Dawley rats (25&300 g) by collagenase perfusion as described by Moldeus et al. (18). The cells were frozen in l-ml samples in Eppendorf tubes at a concentration of 5 X IO6 cells/ml of the cryoprotectant (Nunc cryotubes were used for storage under liquid nitrogen), Each sample of cells was centrifuged at 10,000g (micro-hematocrit centrifuge) for 10 set, the supernatant removed, and the pellet resuspended in the cryoprotectant. Two different cryoprotectant solutions
290
COUNDOURIS
were used. These are referred to as Me,SO cryoprotectant and vitrification solution (VS). VS was based on solutions described by Fahy ei al. (4) and consisted of 20.5% (w/v) Me,SO, 15.5% (w/v) acetamide, 10% (w/v) propylene glycol, and 6% (w/v) polyethylene glycol (relative molecular mass 8000). The cells were stored frozen for up to 7 days either at - 80°C or at - 196°Cin liquid nitrogen. At desired time intervals samples were thawed rapidly at 37°C and washed once with Leibowitz L15 medium, then once with Krebs-Henseleit buffer, pH 7.4, containing 10 mM 4-(d-hydroxyethyl)1-piperazineethanesulfonic acid (Hepes). They were finally resuspended in the same buffer at a concentration of IO x lo6 cells/ ml before their viability was assessed by trypan blue exclusion. Incubation at Hepatocytes The viability and intracellular GSH content of hepatocytes (106/ml) were determined during 37°C incubations under 95% 0,/5% CO2 both pre- and postcryopreservation. Samples were taken from the incubations at timed intervals and the viability and GSH content of the cells measured. Four different incubation media were examined: Krebs-Henseleit buffer containing 10 mM Hepes (KH), KH with 1% (w/v) bovine serum albumin (BSA), Leibowitz L15 medium (L15), and L15 with 1% (w/v) BSA. All media were adjusted to pH 7.4, Analytical Methods Measurements of cytochrome P450, glutathione reductase, and microsomal epoxide hydrolase (EH) were carried out using whole cell homogenates prepared as described by Grant et al. (10). Homogenates were stored for up to 10 days at - 80°C until analysis. The concentration of cytochrome P450 was measured spectrophotometrically (21) using a Varian Cary 219 spectrophotometer. EH was measured by the method of Dansette et al, (2) as modified by Grant
ET AL.
et al. (9) using 20 PM phenanthrene9,10-oxide as the substrate. The enzyme activity was quantified by direct fluorimetric detection of the product phenanthrene9,10-dihydrodiol. The activity of the cytochrome P450-dependent N-demethylation of aminopyrine was determined by measurement of the formaldehyde produced during a IO-min incubation of 20 m&4 aminopyrine with whole cell samples (19). Intracellular GSH concentrations were measured by the method of Saville (24) which measures total reduced thiols, of which 9&95% is GSH. Glutathione reductase activity was measured by following the oxidation of NADPH spectrophotometritally at 340 nm (1). The GSH conjugation of 50 t&f l-chloro,2,4-dinitrobenzene (CDNB) (12) was measured both in whole cells and in cell homogenates. Cells (106/ml) were incubated with 50 FM CDNB at 37°C for up to 30 min and centrifuged at 10,OoOg for 2-3 min, and the absorbance of the supernatant was read immediately at 340 nm. The reaction rate was calculated over the linear pat-t of the time curve. GSH conjugation was also measured in cell homogenates in the presence of 1 mIt4 GSH. The reaction was carried out at 37°C in a cuvette and the increase in absorbance at 340 nm monitored directly. RESULTS
Optimizing the Composition of the Cryoprotectant Media The two cryoprotectant media compared, the Me,SO cryoprotectant and VS, were prepared either in Leibowitz L15 medium or in Dulbecco’s phosphate-buffered saline (PBS) in the presence or absence of 25% (v/v) fetal calf serum (FCS). Cells were frozen in each cryoprotectant medium at 1Wmin to -80°C and stored for 24 hr before being thawed, and cell viability and GSH content were measured. Table 1 shows that the cell viability was maintained significantly better when the
CRYOPRESERVATION
TABLE I Cell Viability and GSH Content after 24 hr Storage at -80°C in 20% (v/v) Me+0 and 25% (v/v) VS (VS25) Cryoprotectants in PBS or Leibowitz L15 Medium Cryoprotectant
Viability
GSH content
Me&i0 in PBS Me,SO in L15 + FCS Me,.50 in L15 ~ FCS
36 (2) 35 f 5 (24) 3123(4)
37 (2) 44 + 9 (4) 40 * 5 (4)
VS25 in PBS
37 + 5 (6) 47 e 10 (4)*q 48 2 6 (%I)*3
40 f 5 (4) 40 + 9 (3) 47 f 8 (12)
VS25
in L15 + FCS
VS25 in LlS - FCS
Note. Cell viability and GSH content after 24 hr storage at - 80°C in Me,SO and 25% VS (VS25) cryoprotectants in phosphate-buffered saline (PBS) or Leibowitz L15 medium in the presence (+) or absence ( -) of 25% (v/v) FCS. Values are means f SEM, with the number of experiments in parentheses, and are expressed as percentages of the initial fresh cell vahes. Fresh cells were 91 + 3% (n = 24) viable and contained 29.6 2 2.0 nmol GSH/106viable cells (n = 14). * P < 0.05 when VSZS is compared with &SO by one-way analysis of variance (significance assigned by Newman-Keuls test). t P < 0.05when VS25 in PBS is compared with VS25 in Leibowitz L15 medium (one-way analysis of variance, significance assigned by Newman-Keulstest).
cells were cryopreserved in (i) 25% (v/v) VS than in 20% (v/v) Me,SO and (ii) when the 25% (v/v) VS was prepared in Leibowitz L15 medium rather than in PBS (P < 0.05). A similar trend was observed for the intracellular GSH content. The addition of FCS (25% v/v) to VS did not improve cell viability postcryopreservation, whereas FCS was required for preserving cell viability and GSH content in the Me,SO cryoprotectant medium (Fig. 1A). The optimal FCS concentration in the Me,SO cryoprotectant was 20-30% (v/v). The optimal Me,SO content in the Me,SO cryoprotectant was found to be 20% (v/v) based on preservation of cell viability and cytochrome P450 and GSH content (Fig. 1B). Figure 2 illustrates that 25% VS was the optimum concentration for maintenance of viability and GSH content in L15 medium. In the remainder of the experiments described in this paper the cryoprotectant media were composed of (i) the Me,SO cryoprotectant [20% (v/v) Me,SO and 25% (v/v) FCS in Leibowitz LI5 medium] and (ii) the VS [25% (v/v) vitrification solution in Lei-
291
OF RAT HEPATOCYTES
I;; 20 I
O4
A
0
tw
B
10
20
s-
30 40 FCS (X, v/v)
50
60
T
0
10
20
30
OMSO (%. v/v1
FIG. 1. Preservation of cell viability (a, n = 3), cytochrome P450 content (A, II = 3), and GSH content (0, n = 3) during 24 hr storage in 20% (v/v) Me,SO with varying FCS concentrations (A) and in 25% (v/v) FCS with varying Me,SO concentrations (B). AU data are means k SEM of three experiments and are expressed as percentages of fresh cell values.
bowitz L15 medium (VS25)]. Cells were thawed as described under Methods. The inclusion of 1M glucose in the first wash, as an attempt to minimize osmotic shock to the cells, resulted in a slight decrease in both cell viability and GSH content. Leibowitz LI5 medium was therefore used without glucose in all subsequent experiments. Optimizing the Freezing Schedule
All sampleswere kept on ice for a period to allow penetration of the cryoprotectant media into the cells before the freezing pro-
292
f &
ET AL.
COUNDOURIS
Ol0 5
15
25 35 vs I%, V/VI
45
56
FIG. 2. Preservation of cell viability f@) and GSH content (0) during 24 hr storage at - 80°C in different VS concentrations in L15 medium. The data are expressed as percentages of the fresh cell values and are means + SEM of three experiments. Fresh cells were 90 + 3% viable (n = 3) and contained 26.0 + 2.0 nmol GSH006 viable cells.
cording to the different freezing schedules listed below and stored at either -80 or - 196°Cfor 24 hr before being thawed for determination of viability and GSH content. For freezing at l”C/min the tubes were placed in the -80°C freezer, whereas for faster freezing rates a programmable freezer (Kryo-10 series, Planar) was used: (i) l”C/min to -80°C; (ii) 5”Clmin to -80°C; (iii) lO”C/min to -80°C; (iv) 2OWmin to -8O”C, then into liquid nitrogen; (v) 3OYYminto - 8O”C,then into liquid nitrogen; (vi) suspending samplesin vapour phase of liquid nitrogen; (vii) plunging samples straight into liquid nitrogen. Table 2 shows that of the freezing schedules examined the optimal one was freezing at a rate of 1”Clmin to -80°C. The lowest cell viability after thawing occurred when the cells were plunged straight into liquid nitrogen. Preservation of Cell Function and Viability after 7 Days Storage at -80°C in Both Cryoprokctants
cedure was started. This equilibration period was varied between 5 and 60 min for each medium and the optimum time for Using the optimal freezing schedule of equilibration was 15 min for VS25 and 10 l”C/min to -80°C and the optimal compomin for the Me&I cryoprotectant (results sition of each medium the period of cryonot shown). The cells were then frozen ac- preservation was extended to 7 days. SamTABLE 2 Effect of Different Freezing Schedules on the Viability and GSH Content of Cells Cryopreserved in Me,SO Cryoprotectant and 25% VS after a 24-hr Storage Period Freezing schedule i ii ..a Ul iv V
vi vii
Me,SO
vs25
Viability
GSH content
Viability
GSH content
34 k 2 (7)* 31 (2) 30 (2)
36 * 2 (7)” 35 (2) 33 (2)
f 4 (6) 38 (21 32 (21 35 f 5 (5)
45 2 3 (4) 39 (21 35 (21 38 f 6 (5)
16(2) 20 G3 18(2)
21 (2) 26 (21 20 (21
25 (2) 1.5 (21 14 (21
10 (2)
31 (2)
22 (2) 21 c4 20 (21
45
Note. The results are means, with the number of experiments in parentheses, and are expressed as percentages of the fresh cell values. Where more than two experiments were carried out the SEM is given. The freezing schedules used were (i) 1Wmin to -80°C; (ii) 5Wmin to -80°C; (iii) IOWti to -80°C; (iv) 2OWrnio to -SO’C, then into liquid nitrogen; (v) 30Wmin to - 8WC, then into liquid nitrogen; (vi) suspension of samples in vapor phase of liquid nitrogen; (vii) plunging of samples straight into liquid nitrogen. * P < 0.05 by nonpaired Student’s I test, when Me,SO medium is compared with VS25.
CRYOPRESERVATION
OF RAT HEPATOCYTES
pies of the cells were thawed after 1,2,3,5, and 7 days, and the cell viability, GSH and cytochrome P450 content, and activities of glutathione+transferase, MFO, epoxide hydrolase, and glutathione reductase were measured. Figure 3 shows the results obtained using the Me,SO cryoprotectant. A slow decrease in cell viability occurred, between 1 day (38 f 2% viable, n = 4) and 7 days (32 f 2% viable, n = 4). Cytochrome P450 content and dependent N-demethylation of aminopyrine were well maintained over the 7-day storage, as was epoxide hydrolase activity. After 7 days the cells contained 97 * 7% (n = 5) of fresh cell cytochrome P450 content and carried out aminopyrine me140 r
s oJ , a 1
,
,
,
3 PRESERVATION
, 5
PERIOD
, 7
(DAYSI
Ftc. 3 Viability (+ , n = 4), cytochrome P450 content (0, n = 5), GSH content (A, n = 4), aminopyrine N-demethylation (A, II = 4), GSSG reductase (W, n = Z), epoxide hydrolase, (m, n = 2), and GSH conjugation with CDNB (0, n = 4) in adult rat hepatocytes during a 7-day storage period at - 80°C in the Me,SO cryoprotectant. Values are means + SEM and are expressed as percentages of the initial fresh cell values. Fresh cell values were viability, 90 f 3% (n = 4); cytochrome P450 content, 0.410 f 0.03 nmoU106cells (n = 5); GSH content, 33.2 k 2.2 nmolGSH1106 viable cells (n = 4); aminopyrine N-demethylation, 15.6 f 2.1 nmoUminil06 cells (n = 4); GSSG reductase, 0.277 ~moknin/106 cells (n = 2); epoxide hydrolase, 1.26 nmol/min/@ cells (n = 2); and CDNB conjugation with GSH, 3.29 k 0.25 nmol/min/@ viable cells.
293
tabolism and epoxide hydrolysis at 111 ? 4% (n = 4) and 120% (n = 2) of that observed in fresh cells, respectively. After 24 hr storage the GSH content had declined to 79 2 3% (n = 4) of fresh cell values and it was maintained at this level throughout the 7-day cryopreservation period. The activity of glutathione-S-transferase toward CDNB declined markedly from 24 hr (78 2 6% of fresh cell values, n = 4) to 7 days (34 + 7%, n = 4) of cryopreservation. This conjugation reaction was carried out in intact cells over 30 min at 37°C. Later experiments showed that thawed cells lost viability rapidly when incubated in suspension at 37°C. The decrease in CDNB conjugation in postcryopreserved cells could, therefore, have been due to loss of intracellular GSH. However, a similar decline in the GSH conjugation of CDNB was observed in homogenates of postcryopreserved hepatocytes in the presence of 1 miW GSH (36 2 4% of the fresh cell values, n = 4, after 7 days). The activity of glutathione reductase declined to 29% (n = 2) of fresh cell values after 24 hr storage at - 80°C and was maintained at this level throughout the 7-day storage period. GSH and cytochrome P450 content and the activities ‘of glutathione-S-transferase, MFO, epoxide hydrolase, and glutathione reductase were similar in VS25 medium over 7 days (data not shown). However, the viability of cells preserved for 24 hr at -80°C was greater in VS25 than in Me,SO cryoprotectant (50 f 2%, n = 4, compared with 38 ? 3%, n = 4, viable in VS25 and Me,SO media, respectively). Following this initial decline in viability after 24 hr, there was a further slow decrease in viability over 7 days storage in both media (Table 3), until after 7 days the cell viability was similar in both cryoprotectant media (32 * 3%, n = 4, viable in Me,SO cryoprotectant compared with 37 + 7%, n = 4, in VS25). This suggests that the VS25 medium may be more toxic to the cells over the 7-day period.
294
COUNDOUBlS
ET AL.
TABLE 3 Viability of Adult Bat Hepatocytes during 7-Day Storage at - 80°C Preservation time WYS) 1 2 3 5 7
Viability Me,SO 38 + 35 + 33 f 30 f 32 k
3 3 4 3 3
VS25 50 + 2 4424 42 2 3 39 * 4 37 k 4
Note. Values are means + SEM (n = 4) and are expressed as percentages of the initial fresh cell values. Fresh cell were 90 + 3% (n = 4) viable.
Viability of Postcryopreserved Suspension at 37°C
Cells in
Thawed hepatocytes were routinely kept on ice before assessment of viability by trypan blue exclusion. The viability and intracellular GSH content of freshly isolated and postcryopreserved (24 hr storage at - 80°C) hepatocytes were monitored at timed intervals at 37°C. The cells were suspended in four different incubation media as described under Methods. Table 4 shows that in all four incubation media the viability of postcryopreserved cells fell rapidly. The rate of decrease in viability was similar in all the media tested. However, after 30 min the viability was higher in the Leibowitz L15 media compared with the KrebsHepes buffers. In addition, cells cryopreserved in 25% VS were better protected from loss of viability than those cryopreserved in Me,SO. In the Krebs-Hepes incubation media intracellular GSH depletion paralleled the rate of cell death. However, in the hepatocytes incubated in Leibowitz L15 medium (in both the presence and the absence of 1% w/v BSA) intracellular GSH levels increased (results now shown), suggesting that some of the synthetic pathways in the cells were still functional despite the loss in viability. To determine the extent to which icecrystal formation was responsible for the decrease in viability postcryopreservation
TABLE 4 Viability of Hepatocytes during a 37°C Incubation Postcryopreservation Incubation medium
Time (min)
KH
0 5 10 20 30
IO0 5726 5125 34 f 5 26 -t 4
0 5 10 20 30
100 65k5 52 f 6 4024 33 + 3
LA
Fresh cells
A. In Me,SO loo 100 55k6 6o-t8 55k4 57?8 32 f 3 41 i 6 24 k 2 35 f 5
loo 6025 52 f 5 40*4 37 + 3
100 95a1 93 f 1 W&2 89 f 3
B. In 100 62+6 53 f 6 35k5 34 f 4
100 7226 69 + 8 54+5 46 + 5
100 %-+I 93 + 2 9121 89 + 3
KA
L15
25% VS 100 8OklI 66 zk 10 46+6 40 + 6
Note. Cells were incubated for varying times in Krebs-Hepes buffer (KH), KH containing 1% (w/v) bovine serum albumin (KA), Leibowitz L15 medium (L15), and Ll5 containing I% (w/v) bovine serum albumin (LA), after a 24-hr cryopreservation period at - 80°C in Me,SO cryoprotectant (A) and 25% VS (B). Values for freshly isolated cells incubated in LA medium are also shown. The values are means + SEM of between three and five experiments. Cell viability is expressed as a percentage of the viability at time 0.
(both before and after 37°C incubations), cells were cooled at a rate of f”C/min to - 10 and - 20°C (above and below the point of ice-crystal formation in the two cryoprotectants). After 15 min and 24 hr storage at these temperatures the cells were thawed rapidly and their viability was measured by trypan blue exclusion (Table 5). They were then incubated for 30 min at 37°C in Leibowitz L15 medium containing 1% (w/v) bovine serum albumin, measuring the viability of the cells at timed intervals (Table 5). The decrease in cell viabihty was less in cells cryopreserved at - 10°C than in cells cryopreserved at -20°C for 15 min. After 24 hr this effect was reversed. However, the decline in cell viability during the 37°C incubations was similar for all three storage temperatures. This would suggest that as well as ice-crystal formation, osmotic and temperature shocks may also contribute to
CRYOPRESERVATION
295
OF RAT HEPATOCYTES
TABLE 5 Postcryopreservation Cell Viability Using Different Freezing Schedules Postcryopreservation cell viability During a 37°C incubation
Freezing schedule
n
Cryoprotectant
At 4°C
5 min
10 min
20 min
30 min
i
4
ii
2
Me,SO VS25 Me,SO vs25 Me,SO VS25 Me,SO VS25 Me,SO vs25
27 + 1 41 + 1 45 55 21 32 28 44 25 39
75 2 3 79 + 5 72 86 76 90 68 71 81 82
68t4 72 k 6 61 67 68 62 64 68 73 67
62 f 5 63 2 2 58 53 39 50 46 46 37 57
38 k 7 47 k 3* 38 48 40 44 38 40 37 42
...
ill
1
iv
2
v
2
Note-The freezing schedules used were (i) 1Wmin to - 8O”C, stored for 24 hr; (ii) l*C/min to - lO”C, stored for 15 min; (iii) 1Wmin to - lO”C, stored for 24 hr; (iv) l”C/min to -2O”C, stored for 15 min; and (v) l”C/min to - 20°C stored for 24 hr. The results are means (where n B Z), with the number of experiments in parentheses. Values at 4°C refer to cell viability immediately after thawing. Cell viability at 4°C is expressed as a percentage of the fresh cell value, and for the 37°C incubation, as a percentage of the viability at time 0. * P < 0.05, when Me,SO medium is compared with VS25 medium by nonpaired Student’s t test.
the loss in cell viability during cryopreservation. To determine whether the rapid loss of viability during 37°C incubations was due to oxidation of essential cofactors or to protein degradation, 5 miI4 dithiothreitol (an antioxidant), 0.25 mM leupeptin (protease inhibitor), and 10 m&I ammonium chloride (inhibitor of lysosomal protease activity) were added individually and in combination to the washing buffers and the incubation mixtures, but no improvement in the maintenance of cell viability was observed (results not shown). Frim and Mazur (5) have suggested that the rapid loss in the viability of granulocytes incubated at 37°C postcryopreservation is due to the high lysosomal content of the cells, but this does not appear to be the case for hepatocytes. Hepatocytes were examined by transmission electron microscopy and Fig. 4 shows the typical appearance of the majority of the hepatocytes when freshly isolated (A); after 24 hr cryopreservation at -80°C followed by thawing and maintenance on ice (B), and after 30 min incubation of postcryopreserved (24 hr at -8O’C) cells at 37°C
(C). Table 6 categorizes the types of cell damage typically observed in the cells and the proportion of “normal” hepatocytes present in a sample of 100 cells examined in Figs. 4A-C. In the fresh cell samples, the majority of the cells, 65%, are normal. However, postcryopreservation, at 4°C only 28% of the ceils appeared normal, 21% showed major internal damage, and 32% showed additional partial loss of plasmalemma. After 37°C incubation, there were no structurally normal cells; most showed severe damage with partial or complete loss of plasmalemma (44 and 24% of the cells, respectively). The electron microscopic appearance of the cells postcryopreservation at 4°C was better than that described by Fuller et al. (7), who found only 10% normal cells after freezing to -60°C. DISCUSSION
For the cryopreservation of rat hepatocytes, the optimum composition of the Me,SO cryoprotectant was found to be 20% (v/v) Me,SO and 25% (v/v) FCS in Leibowitz L15 medium and for the VS, 25% (v/v) of the vitrification solution in Leibowitz L15
296
COUNDOURIS
ET AL.
0A
/ ” N
. f’
-
. .
CRYOPRESERVATION
OF RAT HEPATOCYTES
297
FIG. 4. Electron micrographs showing (A) the normal appearance of freshly isolated hepatocytes; (B) cytoplasmic swelling with lucency of organelles and glycogen clumping after 24 hr cryopreservation at -SOT, kept on ice; and (C) gross cytoplasmic and nuclear damage with plasmalemmal loss postcryopreservation and after a 37°C incubation for 30 min. Bar = 5 Frn.
medium. Freezing at a rate of l”C/min down to -80°C was the optimum freezing schedule for both cryoprotectant media. As previously observed by Fuller et al. (8) viability after thawing was lowest when the cells were plunged straight into liquid nitrogen, i.e., cooled rapidly to - 196°C directly from 4°C (20&400°C/min). Rat hepatocytes were cryopreserved for up to 7 days after freezing at 1”Clmin down to -80°C. During this period the cytochrome P450 content of the cells and the P450-dependent N-demethylation of aminopyrine were maintained at almost 100%. In fact, the N-demethylation activity of postcryopreserved cells was higher than that obtained for fresh cells (see Fig. 3), suggesting that a specific subpopulation of he-
patocytes with higher MFO activity may have survived the cryopreservation process. Previous workers have also found that the cytochrome P450-dependent metabolism of aldrin and 7-ethoxycoumarin in postcryopreserved hepatocytes is greater than in freshly isolated cells (25). Freezing at a rate of 1”Clmin down to - 80°C also resulted in improved maintenance of cytochrome P450 compared with a much more complex freezing schedule to - 196°C in a medium containing 10% Me,SO (22). In contrast to the activities of the microsomal MFO and epoxide hydrolase enzymes, glutathione-S-transferase and glutathione reductase activities fell sharply after cryopreservation. Glutathione-S-transferase activity declined to 78 f 6% of fresh
298
COUNDOURIS TABLE
6
Postcryopreservation cells (%) Cell Type
Fresh cells m
At 4°C
After 30 min at 37°C
1 2 3 4 5
65 9 17 4 4
28 4 21 32 15
0 22 10 44 24
Debris
k
+
++
Note. Rat hepatocytes, freshly isolated and after 24 hr cryopreservation in the Me,SO cryoprotectant at -WC, were examined by electron microscopy. Cryopreserved cells were either kept on ice (4°C) or incubated at 37°C in Krebs-Henseleit buffer containing 1% (w/v) bovine serum albumin for 30 min. The cells were then classified into five types depending on their morphology: (1) normal; (2) blebs on cell surface and/or clumped glycogen; (3) as 2 but with increased lucency of cytosol and organelles, dilated endoplastnic reticulum + cell swelling; (4) as 3 but more cell swelling and partial loss of plasmalemma; (5) as 4 but with total loss of plasmalemma although nucleus and cytosol still “aggregated”, the mitochondria appear swollen. Debris = isolated cell structures not in obvious “aggregates.”
cell values after 24 hr storage at - 80°C and to 34 + 7% after 7 days. The glutathione reductase activity was only 29% of fresh cell activity after 24 hr and remained at this low level throughout the 7 days. These results suggest a differential stability of the microsomal and cytosolic enzymes of hepatocytes after cryopreservation. Cytosolic enzymes may be more exposed to icecrystal damage, whereas the microsomal membrane may offer some protection to the enzymes embedded in it. These results are supported by a recent study by Powis et al. (22), where sulfotransferase activity was lost to an appreciably greater extent than UDP-glucuronosyl transferase activity in cryopreserved rat hepatocytes with biphenyl as substrate. A substantial fall in cell viability was observed after 24 hr cryopreservation at - 80°C for both cryoprotectant media used,
ET AL.
this fall being more marked for the Me,SO cryoprotectant. This initial rapid fall in viability was followed by a further slow decrease in viability over the 7-day cryopreservation period, suggesting that the actual freezing-thawing process causes greater damage to hepatocytes than does the length of the cryopreservation storage period. The gradual fall in viability over 7 days is greater in VS25 cryoprotectant, and this suggests that the vitrification solution may be more toxic to the cells. Although no significant improvement in viability was noted when the cells were stored in liquid nitrogen, it is possible that a slower freezing rate to -80°C and subsequent storage in liquid nitrogen may be beneficial for longer-term storage. A sharp fall in viability was observed during 30-min incubations at 37°C of the hepatocytes postcryopreservation; this may be partly responsible for the low MFO activities reported after cryopreservation (22), if incubation periods in excess of 10 min are used. The loss of viability is unlikely to be due to oxidative stress, as the fall in intraceilular GSH paralleled, rather than preceded, cell death. Cells cryopreserved in 25% (v/v) VS and incubated at 37°C in Leibowitz LI5 medium containing 1% (w/v) BSA showed the best maintenance of viability during the incubation (44% viable after 30 min, taking the cell viability at time 0 as 100%). This could be due to the synthesis of GSH in Leibowitz L15 medium, although Fuller et al. (8) have reported that protein synthesis is only 25% of control values, after thawing of cells frozen down to - 196°C. Karlberg and LindahlKiessling (15), describing only two samples, found no marked loss of viability in cells incubated for 1 hr at 37°C in RPM1 1640 containing 1% bovine serum albumin, after storage in liquid nitrogen. The 37°C incubation was carried out at a lower cell density, which could be responsible for the improved viability. The problem of the rapid loss in postcry-
CRYOPRESERVATION
OF RAT HEPATOCYTES
opreservation cell viability at 37°C in suspension along with the sharp decline in the activities of cytosolic enzymes during cryopreservation will have to be overcome before cryopreserved hepatocytes can be used as a model for studying drug metabolism and toxicity. However, we have promising initial results to indicate that cryopreserved rat and human adult hepatocytes attach and survive in primary culture without the rapid loss in viability observed in suspensions of cells at 37°C. The attachment of cells to the collagen-coated culture dishes may help to stabilize the cell membranes. The survival of an atypical population of hepatocytes with higher MFO activities than normal will continue to be a problem. ACKNOWLEDGMENTS We acknowledge the support of the Humane Research Trust for a Ph.D. studentship (J.A.C.), the Wellcome Trust (M.H.G.), the Scottish Home and Health Department, and the Grampian Health Board (for materials). REFERENCES 1. Carlberg, I., and Manner& B. Glutathione reductase. In “Methods in Enzymology” (A. Meister, Ed.), Vol. 113, pp. 484-490. Academic Press, New York, 1987. 2. Dansette, P. M., DuBois, G. C., and Jerina, D. M. Continuous tluorometric assay of epoxide hydrase activity. Anal. Biochem. 97,34& 345 (19791. 3. Dansette, P. M., and Jerina, D. M. A facile synthesis of arene oxides at the K regions of polycyclic hydrocarbons. J. Amer. Chem. Sot. 96, 122&1225 (1974). 4. Fahy, G. M., McFarlane, D. R., Angell, C. A., and Meryman, H. T. Vitrification as an approach to cryopreservation. Cryobidogy 21, 407426 (1984). 5. Frim, J., and Mazur, P. Interactions of cooling rate, warming rate, glycerol concentration and dilution procedure on the viability of frozenthawed human granulocytes. Cryobiology 20, 657-676 (1983). 6. Fry, J. R., and Bridges, J. W. Use of primary hepatocyte cultures in biochemical toxicology. In “Reviews in Biochemical Toxicology” (E. Hodgson, J. R. Bend, and R. M. Philpot, Eds.), Vol. 1, pp. 201-247. Elsevier/North-Holland, Amsterdam, 1979.
299
7. Fuller, B. J., Grout, B. W., and Woods, R. J. Biochemical and ultrastructural examination of cryopreserved hepatocytes in rats. Cryobiology 19,493-502 (1982). 8. Fuller, B. J., Morris, G. J., Nutt, L. H., and Attenburrow, V. D. Functional recovery of isolated rat hepatocytes upon thawing from - l%“C. Cryu-Letters 1, 139-146 (1980). 9. Grant, M. H., Burke, M. D., Hawksworth, G. M., Duthie, S. J., Engeset, J., and Petrie, J. C. Human adult hepatocytes in primary monolayer culture: Maintenance of mixed function oxidase and conjugation pathways of drug metabolism. Biochem. Pharmacof. 36, 231l2316 (1987). 10. Grant, M. H., Melvin, M. A. L., Shaw, P., Melvin, W. T., and Burke, M. D. Studies on the maintenance of cytochromes P-450 and bg, monooxygenase and cytochrome reductase in primary cultures of rat hepatocytes. FEES Lett. 190, 99 (1985). 11. Guillouzo, A., Guguen-Guillouzo, C., and Bourel, M. Hepatocytes in culture: Expression of differentiated functions and their application in the study of metabolism. Triangle 20, 121-128 (1981). 12. Habig, W. H., and Jakoby, W. B. Assays for differentiation of glutaihione-S-transferase. In “Methods in Enzymology” (W. B. Jakoby, Ed.), Vol. 77, pp. 298-405. Academic Press, New York, 1981. 13. Holme, J. A., Soderlund, E., andDybing, E. Drug metabolism activities of isolated rat hepatocytes in monolayer culture. Acfa Pharmacol. Toxicol. 52, 348-356 (1983). 14. Kapitulnik, J., Wislocki, P. G., Levin, W., Yagi, H., Jerina, D. M., and Conney, A. H. Tumorigenicity studies with dial-epoxides of benzo(a)-pyrene which indicate that (+)-trans-Q,8cc, lOa-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene is an ultimate carcinogen in newborn mice. Cancer Res. 38, 354-358 (1978). 15. Karlberg, I., and Lindahl-Kiessling, K. Preservation of freshly isolated liver cells in liquid nitrogen at - 196°C.Mutat. Res. 85,411416 (1981). 16. Le Cam, A., Guillouzo, A., and Freychet, P. UItrastructural and biochemical studies of isolated adult rat hepatocytes prepared under hypoxic conditions. Cryopreservation of hepatocytes. Exp. Cell Res. 98, 382-395 (1976). 17. Levin, W., Thakker, D. R., Wood, A. W., Chang, R. L., Lebr, R. E., Jerina, D. M., and Conney, A. H. Evidence that benzo(a)anthracene 3,4diol-1,2-epoxide is an ultimate carcinogen on mouse skin. Cancer Rex. 38, 1705-1710 (1978). IS. Moldeus, P., Hogberg, J., and Orrenius, S. Isolation and use of liver cells. In “Methods in En-
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zymology” (S. Pleischer and L. Packer, Eds), Vol. 52, pp. 60-71. Academic Press, New York, 1978. 19. Nash, T. The calorimetric estimation of formaldehyde by means of the Hantzsh reaction. Biothem. J. 55,41U21 (1953). 20. Novicki, D. L., Irons, 0. P., Strom, S. C., Jirtle, P., and Michalopoulos, G. Cryopreservation of isolated rat hepatocytes. In Vitro 18, 393-399 (1982). 21. Omura, T., and Sato, R. Carbon-monoxidebinding pigment of liver microsomes. I. Evidence for its haemoprotein nature. J. Bid. Chem. 239,237s2385 (1964). 22. Powis, G., Santone, K. S., Melder, L., Moore, D. J., and Wilke, T. J. Cryopreservation of rat and dog hepatocytes of studies of xenobiotic metabolism and activation. Drug Metab. Dispm. 15, 826-832 (1987).
23. Reed, D. J. Regulation of reductive processes by glutathione. Eiochem. Pharmacol. 35, 7-13 (1986). 24. Saville, B. A scheme for the calorimetric determination of microgram amounts of thiols. Analyst 83, 670-672 (1958). 25. Seddon, T., Boobis, A. R., and Davies, D. S. Drug metabolising activity of cryopreserved rat and human hepatocytes. &it. J. Clin. Pharmacd. 20, 546P (1985). 26. Strom, S. C., Jirtle, R. L., Jones, R. S., Novicki, D. L., Rosenberg, M. R., Novotny, A., Irons, G., McLain, J. R., and Michalopoulos, G. Isolation, culture and transplantation of human hepatocytes. .I. NatI. Cancer Inst. 68, 771-778 (1982). 27. Walker, C. H. Species differences in microsomal monooxygenase activity and their relationship to biological half-lives. Drug Metab. Rev. 7, 295-323 (1978).
27, 288-300
(l%‘tl)
Drug Metabolism
and Viability Studies Rat Hepatocytes
in Cryopreserved
J. A. COUNDOURIS,**t M. H. GRANT,* J. G. SIMPSON,S AND G. M. HAWKSWORTH*?t Departments of *Medicine and Therapeutics (Unit qf Clinical Pharmacology), fPharmaco/ogy, and #Pathology, University of Aberdeen, Polwarth Building, Foresterhill, Aberdeen AB9 220, United Kingdom Rat hepatocytes were cryopreserved optimalty by freezing them at 1Wmin to - 80°C in cryoprotectant medium containing either 20% (v/v) diiethylsulfoxide (Me,SO) and 25% (v/v) fetal calf serum in Leibowitz LlS medium (Me,!30 cryoprotectant) or 25% (v/v) vitrification solution (containing Me,SO, acetamide, propylene glycol and polyethylene glycol) in Leibowitz Ll5 medium (VS25). The VS25 solution was superior for maintaining viability during short-term storage (2W hr) but was slightly toxic during longer storage periods (7 days). Although thawed cells were 40-K% viable on ice after cryopreservation, their viability fell rapidly during incubation in suspension at 37°C. This decline in viability occurred more rapidly after freezing in Me,SO cryoprotectant than in VS25 and was associated with extensive intracellular damage and cell swelling. The loss in viability at 37°C does not appear to be due to ice-crystal damage as it occurred in cells stored at - 10°C{above the freezing point of the cryoprotectants) and it may be due to temperature/osmotic shock. Both cryoprotectant media were equally efficient at preserving enzyme activities in the hepatocytes over 7 days at -80°C. Cytochrome P450 and reduced glutathione content and the activities of the microsomal enzymes responsible for aminopyrine N-demethylation and epoxide hydrolysis were well maintained over 7 days storage. In contrast, the cytosolic enzymes glutathione-S-transferase and glutathione reductase were markedly labile during cryopreservation. Cytosolic enzymes may be more susceptible to icecrystal damage, whereas the microsomal membrane may protect the enzymes which are embedded in it.
0 1990 Academic
Press, Inc.
The metabolism and cytotoxic potential of xenobiotics in humans are usually estimated by extrapolation from animal studies. However, there is evidence that marked species differences in drug metabolism exist (27). It is therefore essential to develop systems that can be used to measure the metabolism and predict the toxicity of xenobiotics in humans. Suspensions of human liver cells would provide a defined system for this work as they contain the enzymes responsible for xenobiotic metabolism and simultaneously provide a target cell for the assessment of toxicity. Viable human adult hepatocytes can be isolated by perfusion of liver segments obtained from kidney donor patients (26); liver obtained postmortem is unsuitable for this purpose. However, the supply Received June 9, 1988; accepted July 27, 1989. 288 0011-2240190 Copyright All rights
$3.00
0 IS90 by Academic Pre66, Inc. of reproduction in any form reserved.
of human liver is infrequent and unpredictable. When suitable material is obtained, large numbers (600 x 10”) of hepatocytes are available in suspension but they remain viable for only about 4 hr. Therefore, one of the most urgent practical requirements is the ability to prolong the useful lifetime of these hepatocyte preparations. The approach most frequently used to prolong the lifetime of hepatocytes is primary culture. A major problem inherent in the culture of mammalian hepatocytes is the instability of a number of specific drugmetabolizing enzymes (11). For example, cytochrome P450-dependent mixed function oxidase (MFO) activity declines to low levels within the first 2&8 hr of culture (6, 13). Although the drug-metabolizing enzyme activities of human hepatocytes are more stable during culture than those of rodent hepatocytes, the ratios of MFO activ-
CRYOPRESERVATION
289
OF RAT HEPATOCYTES
ities and conjugation reactions are continuously changing (9). We have, therefore, sought an alternative method of prolonging the useful lifetime of hepatocytes by cryopreservation of the cells. This process has been used previously for the preservation of human oocytes, sperm, embryos, and other cell types. Previous attempts to cryopreserve hepatocytes have shown that dimethylsulfoxide (Me,SO) is the most suitable cryoprotectant available (8, 16, 20). Many xenobiotics are activated to reactive metabolites by the MFO system and detoxified by various conjugation reactions including conjugation with reduced glutathioine (GSH) and glucuronic acid. The extent of cytotoxicity depends on the balance between these activation and detoxification processes. For cryopreserved hepatocytes to act as a valid system for toxicity studies, they must, after thawing, maintain all the activation and detoxification reactions of drug metabolism at levels found in freshly isolated hepatocytes. In this paper we have compared the ability of two Me,SO-based cryoprotectant media to preserve the viability and drugmetabolizing activity of isolated rat hepatocytes. To assess the preservation of the MFO system, the cellular cytochrome P450 content and the N-demethylation of aminopyrine were measured in the hepatocytes postcryopreservation. Intracellular glutathione participates in such diverse biological processes as detoxification of xenobiotits, removal of hydroperoxides, maintenance of sulfhydryl status of proteins, and modulation of enzyme activities by sulfhydry1 exchange. In cells, glutathione is effectively maintained in the reduced state by glutathione reductase, which is linked to the NADPH/NADP+ system, and intracellular GSH homeostasis exerts a major influence on the status of the NADPH pool (23). Measurement of intracellular GSH levels, GSH-S-transferase, and glutathione reductase activities in postcryopreserved hepatocytes gives an indication of their ability to
protect the cells from xenobiotic-induced cytotoxicity. Many cytotoxic and carcinogenic compounds form reactive epoxides (14, 17) which are subsequently detoxified by epoxide hydrolase. We have measured the activity of the microsomal epoxide hydrolase as a further indication of the metabolic competence of the cryopreserved hepatocytes. These studies will subsequently be extended to human hepatocytes once the optimum cryopreservation conditions have been developed. MATERIALS
Isocitric acid, isocitrate dehydrogenase, bovine serum albumin (fraction V), polyethylene glycol (relative molecular mass 8000), Me,SO, NADPH, and reduced glutathione were all obtained from Sigma Chemical Company (Poole, Dorset). NADP and collagenase were from BoehringerMannheim and acetamide and propylene glycol from BDH Ltd. Fetal calf serum was obtained from Gibco and the Leibowitz L15 medium from Flow Laboratories. Phenanthrene-9, lo-oxide and phenanthrene-9, IOdihydrodiol were prepared as previously described (3). METHODS
Preparation, Freezing, of Hepatocytes
and Thawing
Adult rat hepatocytes (viability > 85%, assessed by trypan blue exclusion) were prepared from male Sprague-Dawley rats (25&300 g) by collagenase perfusion as described by Moldeus et al. (18). The cells were frozen in l-ml samples in Eppendorf tubes at a concentration of 5 X IO6 cells/ml of the cryoprotectant (Nunc cryotubes were used for storage under liquid nitrogen), Each sample of cells was centrifuged at 10,000g (micro-hematocrit centrifuge) for 10 set, the supernatant removed, and the pellet resuspended in the cryoprotectant. Two different cryoprotectant solutions
290
COUNDOURIS
were used. These are referred to as Me,SO cryoprotectant and vitrification solution (VS). VS was based on solutions described by Fahy ei al. (4) and consisted of 20.5% (w/v) Me,SO, 15.5% (w/v) acetamide, 10% (w/v) propylene glycol, and 6% (w/v) polyethylene glycol (relative molecular mass 8000). The cells were stored frozen for up to 7 days either at - 80°C or at - 196°Cin liquid nitrogen. At desired time intervals samples were thawed rapidly at 37°C and washed once with Leibowitz L15 medium, then once with Krebs-Henseleit buffer, pH 7.4, containing 10 mM 4-(d-hydroxyethyl)1-piperazineethanesulfonic acid (Hepes). They were finally resuspended in the same buffer at a concentration of IO x lo6 cells/ ml before their viability was assessed by trypan blue exclusion. Incubation at Hepatocytes The viability and intracellular GSH content of hepatocytes (106/ml) were determined during 37°C incubations under 95% 0,/5% CO2 both pre- and postcryopreservation. Samples were taken from the incubations at timed intervals and the viability and GSH content of the cells measured. Four different incubation media were examined: Krebs-Henseleit buffer containing 10 mM Hepes (KH), KH with 1% (w/v) bovine serum albumin (BSA), Leibowitz L15 medium (L15), and L15 with 1% (w/v) BSA. All media were adjusted to pH 7.4, Analytical Methods Measurements of cytochrome P450, glutathione reductase, and microsomal epoxide hydrolase (EH) were carried out using whole cell homogenates prepared as described by Grant et al. (10). Homogenates were stored for up to 10 days at - 80°C until analysis. The concentration of cytochrome P450 was measured spectrophotometrically (21) using a Varian Cary 219 spectrophotometer. EH was measured by the method of Dansette et al, (2) as modified by Grant
ET AL.
et al. (9) using 20 PM phenanthrene9,10-oxide as the substrate. The enzyme activity was quantified by direct fluorimetric detection of the product phenanthrene9,10-dihydrodiol. The activity of the cytochrome P450-dependent N-demethylation of aminopyrine was determined by measurement of the formaldehyde produced during a IO-min incubation of 20 m&4 aminopyrine with whole cell samples (19). Intracellular GSH concentrations were measured by the method of Saville (24) which measures total reduced thiols, of which 9&95% is GSH. Glutathione reductase activity was measured by following the oxidation of NADPH spectrophotometritally at 340 nm (1). The GSH conjugation of 50 t&f l-chloro,2,4-dinitrobenzene (CDNB) (12) was measured both in whole cells and in cell homogenates. Cells (106/ml) were incubated with 50 FM CDNB at 37°C for up to 30 min and centrifuged at 10,OoOg for 2-3 min, and the absorbance of the supernatant was read immediately at 340 nm. The reaction rate was calculated over the linear pat-t of the time curve. GSH conjugation was also measured in cell homogenates in the presence of 1 mIt4 GSH. The reaction was carried out at 37°C in a cuvette and the increase in absorbance at 340 nm monitored directly. RESULTS
Optimizing the Composition of the Cryoprotectant Media The two cryoprotectant media compared, the Me,SO cryoprotectant and VS, were prepared either in Leibowitz L15 medium or in Dulbecco’s phosphate-buffered saline (PBS) in the presence or absence of 25% (v/v) fetal calf serum (FCS). Cells were frozen in each cryoprotectant medium at 1Wmin to -80°C and stored for 24 hr before being thawed, and cell viability and GSH content were measured. Table 1 shows that the cell viability was maintained significantly better when the
CRYOPRESERVATION
TABLE I Cell Viability and GSH Content after 24 hr Storage at -80°C in 20% (v/v) Me+0 and 25% (v/v) VS (VS25) Cryoprotectants in PBS or Leibowitz L15 Medium Cryoprotectant
Viability
GSH content
Me&i0 in PBS Me,SO in L15 + FCS Me,.50 in L15 ~ FCS
36 (2) 35 f 5 (24) 3123(4)
37 (2) 44 + 9 (4) 40 * 5 (4)
VS25 in PBS
37 + 5 (6) 47 e 10 (4)*q 48 2 6 (%I)*3
40 f 5 (4) 40 + 9 (3) 47 f 8 (12)
VS25
in L15 + FCS
VS25 in LlS - FCS
Note. Cell viability and GSH content after 24 hr storage at - 80°C in Me,SO and 25% VS (VS25) cryoprotectants in phosphate-buffered saline (PBS) or Leibowitz L15 medium in the presence (+) or absence ( -) of 25% (v/v) FCS. Values are means f SEM, with the number of experiments in parentheses, and are expressed as percentages of the initial fresh cell vahes. Fresh cells were 91 + 3% (n = 24) viable and contained 29.6 2 2.0 nmol GSH/106viable cells (n = 14). * P < 0.05 when VSZS is compared with &SO by one-way analysis of variance (significance assigned by Newman-Keuls test). t P < 0.05when VS25 in PBS is compared with VS25 in Leibowitz L15 medium (one-way analysis of variance, significance assigned by Newman-Keulstest).
cells were cryopreserved in (i) 25% (v/v) VS than in 20% (v/v) Me,SO and (ii) when the 25% (v/v) VS was prepared in Leibowitz L15 medium rather than in PBS (P < 0.05). A similar trend was observed for the intracellular GSH content. The addition of FCS (25% v/v) to VS did not improve cell viability postcryopreservation, whereas FCS was required for preserving cell viability and GSH content in the Me,SO cryoprotectant medium (Fig. 1A). The optimal FCS concentration in the Me,SO cryoprotectant was 20-30% (v/v). The optimal Me,SO content in the Me,SO cryoprotectant was found to be 20% (v/v) based on preservation of cell viability and cytochrome P450 and GSH content (Fig. 1B). Figure 2 illustrates that 25% VS was the optimum concentration for maintenance of viability and GSH content in L15 medium. In the remainder of the experiments described in this paper the cryoprotectant media were composed of (i) the Me,SO cryoprotectant [20% (v/v) Me,SO and 25% (v/v) FCS in Leibowitz LI5 medium] and (ii) the VS [25% (v/v) vitrification solution in Lei-
291
OF RAT HEPATOCYTES
I;; 20 I
O4
A
0
tw
B
10
20
s-
30 40 FCS (X, v/v)
50
60
T
0
10
20
30
OMSO (%. v/v1
FIG. 1. Preservation of cell viability (a, n = 3), cytochrome P450 content (A, II = 3), and GSH content (0, n = 3) during 24 hr storage in 20% (v/v) Me,SO with varying FCS concentrations (A) and in 25% (v/v) FCS with varying Me,SO concentrations (B). AU data are means k SEM of three experiments and are expressed as percentages of fresh cell values.
bowitz L15 medium (VS25)]. Cells were thawed as described under Methods. The inclusion of 1M glucose in the first wash, as an attempt to minimize osmotic shock to the cells, resulted in a slight decrease in both cell viability and GSH content. Leibowitz LI5 medium was therefore used without glucose in all subsequent experiments. Optimizing the Freezing Schedule
All sampleswere kept on ice for a period to allow penetration of the cryoprotectant media into the cells before the freezing pro-
292
f &
ET AL.
COUNDOURIS
Ol0 5
15
25 35 vs I%, V/VI
45
56
FIG. 2. Preservation of cell viability f@) and GSH content (0) during 24 hr storage at - 80°C in different VS concentrations in L15 medium. The data are expressed as percentages of the fresh cell values and are means + SEM of three experiments. Fresh cells were 90 + 3% viable (n = 3) and contained 26.0 + 2.0 nmol GSH006 viable cells.
cording to the different freezing schedules listed below and stored at either -80 or - 196°Cfor 24 hr before being thawed for determination of viability and GSH content. For freezing at l”C/min the tubes were placed in the -80°C freezer, whereas for faster freezing rates a programmable freezer (Kryo-10 series, Planar) was used: (i) l”C/min to -80°C; (ii) 5”Clmin to -80°C; (iii) lO”C/min to -80°C; (iv) 2OWmin to -8O”C, then into liquid nitrogen; (v) 3OYYminto - 8O”C,then into liquid nitrogen; (vi) suspending samplesin vapour phase of liquid nitrogen; (vii) plunging samples straight into liquid nitrogen. Table 2 shows that of the freezing schedules examined the optimal one was freezing at a rate of 1”Clmin to -80°C. The lowest cell viability after thawing occurred when the cells were plunged straight into liquid nitrogen. Preservation of Cell Function and Viability after 7 Days Storage at -80°C in Both Cryoprokctants
cedure was started. This equilibration period was varied between 5 and 60 min for each medium and the optimum time for Using the optimal freezing schedule of equilibration was 15 min for VS25 and 10 l”C/min to -80°C and the optimal compomin for the Me&I cryoprotectant (results sition of each medium the period of cryonot shown). The cells were then frozen ac- preservation was extended to 7 days. SamTABLE 2 Effect of Different Freezing Schedules on the Viability and GSH Content of Cells Cryopreserved in Me,SO Cryoprotectant and 25% VS after a 24-hr Storage Period Freezing schedule i ii ..a Ul iv V
vi vii
Me,SO
vs25
Viability
GSH content
Viability
GSH content
34 k 2 (7)* 31 (2) 30 (2)
36 * 2 (7)” 35 (2) 33 (2)
f 4 (6) 38 (21 32 (21 35 f 5 (5)
45 2 3 (4) 39 (21 35 (21 38 f 6 (5)
16(2) 20 G3 18(2)
21 (2) 26 (21 20 (21
25 (2) 1.5 (21 14 (21
10 (2)
31 (2)
22 (2) 21 c4 20 (21
45
Note. The results are means, with the number of experiments in parentheses, and are expressed as percentages of the fresh cell values. Where more than two experiments were carried out the SEM is given. The freezing schedules used were (i) 1Wmin to -80°C; (ii) 5Wmin to -80°C; (iii) IOWti to -80°C; (iv) 2OWrnio to -SO’C, then into liquid nitrogen; (v) 30Wmin to - 8WC, then into liquid nitrogen; (vi) suspension of samples in vapor phase of liquid nitrogen; (vii) plunging of samples straight into liquid nitrogen. * P < 0.05 by nonpaired Student’s I test, when Me,SO medium is compared with VS25.
CRYOPRESERVATION
OF RAT HEPATOCYTES
pies of the cells were thawed after 1,2,3,5, and 7 days, and the cell viability, GSH and cytochrome P450 content, and activities of glutathione+transferase, MFO, epoxide hydrolase, and glutathione reductase were measured. Figure 3 shows the results obtained using the Me,SO cryoprotectant. A slow decrease in cell viability occurred, between 1 day (38 f 2% viable, n = 4) and 7 days (32 f 2% viable, n = 4). Cytochrome P450 content and dependent N-demethylation of aminopyrine were well maintained over the 7-day storage, as was epoxide hydrolase activity. After 7 days the cells contained 97 * 7% (n = 5) of fresh cell cytochrome P450 content and carried out aminopyrine me140 r
s oJ , a 1
,
,
,
3 PRESERVATION
, 5
PERIOD
, 7
(DAYSI
Ftc. 3 Viability (+ , n = 4), cytochrome P450 content (0, n = 5), GSH content (A, n = 4), aminopyrine N-demethylation (A, II = 4), GSSG reductase (W, n = Z), epoxide hydrolase, (m, n = 2), and GSH conjugation with CDNB (0, n = 4) in adult rat hepatocytes during a 7-day storage period at - 80°C in the Me,SO cryoprotectant. Values are means + SEM and are expressed as percentages of the initial fresh cell values. Fresh cell values were viability, 90 f 3% (n = 4); cytochrome P450 content, 0.410 f 0.03 nmoU106cells (n = 5); GSH content, 33.2 k 2.2 nmolGSH1106 viable cells (n = 4); aminopyrine N-demethylation, 15.6 f 2.1 nmoUminil06 cells (n = 4); GSSG reductase, 0.277 ~moknin/106 cells (n = 2); epoxide hydrolase, 1.26 nmol/min/@ cells (n = 2); and CDNB conjugation with GSH, 3.29 k 0.25 nmol/min/@ viable cells.
293
tabolism and epoxide hydrolysis at 111 ? 4% (n = 4) and 120% (n = 2) of that observed in fresh cells, respectively. After 24 hr storage the GSH content had declined to 79 2 3% (n = 4) of fresh cell values and it was maintained at this level throughout the 7-day cryopreservation period. The activity of glutathione-S-transferase toward CDNB declined markedly from 24 hr (78 2 6% of fresh cell values, n = 4) to 7 days (34 + 7%, n = 4) of cryopreservation. This conjugation reaction was carried out in intact cells over 30 min at 37°C. Later experiments showed that thawed cells lost viability rapidly when incubated in suspension at 37°C. The decrease in CDNB conjugation in postcryopreserved cells could, therefore, have been due to loss of intracellular GSH. However, a similar decline in the GSH conjugation of CDNB was observed in homogenates of postcryopreserved hepatocytes in the presence of 1 miW GSH (36 2 4% of the fresh cell values, n = 4, after 7 days). The activity of glutathione reductase declined to 29% (n = 2) of fresh cell values after 24 hr storage at - 80°C and was maintained at this level throughout the 7-day storage period. GSH and cytochrome P450 content and the activities ‘of glutathione-S-transferase, MFO, epoxide hydrolase, and glutathione reductase were similar in VS25 medium over 7 days (data not shown). However, the viability of cells preserved for 24 hr at -80°C was greater in VS25 than in Me,SO cryoprotectant (50 f 2%, n = 4, compared with 38 ? 3%, n = 4, viable in VS25 and Me,SO media, respectively). Following this initial decline in viability after 24 hr, there was a further slow decrease in viability over 7 days storage in both media (Table 3), until after 7 days the cell viability was similar in both cryoprotectant media (32 * 3%, n = 4, viable in Me,SO cryoprotectant compared with 37 + 7%, n = 4, in VS25). This suggests that the VS25 medium may be more toxic to the cells over the 7-day period.
294
COUNDOUBlS
ET AL.
TABLE 3 Viability of Adult Bat Hepatocytes during 7-Day Storage at - 80°C Preservation time WYS) 1 2 3 5 7
Viability Me,SO 38 + 35 + 33 f 30 f 32 k
3 3 4 3 3
VS25 50 + 2 4424 42 2 3 39 * 4 37 k 4
Note. Values are means + SEM (n = 4) and are expressed as percentages of the initial fresh cell values. Fresh cell were 90 + 3% (n = 4) viable.
Viability of Postcryopreserved Suspension at 37°C
Cells in
Thawed hepatocytes were routinely kept on ice before assessment of viability by trypan blue exclusion. The viability and intracellular GSH content of freshly isolated and postcryopreserved (24 hr storage at - 80°C) hepatocytes were monitored at timed intervals at 37°C. The cells were suspended in four different incubation media as described under Methods. Table 4 shows that in all four incubation media the viability of postcryopreserved cells fell rapidly. The rate of decrease in viability was similar in all the media tested. However, after 30 min the viability was higher in the Leibowitz L15 media compared with the KrebsHepes buffers. In addition, cells cryopreserved in 25% VS were better protected from loss of viability than those cryopreserved in Me,SO. In the Krebs-Hepes incubation media intracellular GSH depletion paralleled the rate of cell death. However, in the hepatocytes incubated in Leibowitz L15 medium (in both the presence and the absence of 1% w/v BSA) intracellular GSH levels increased (results now shown), suggesting that some of the synthetic pathways in the cells were still functional despite the loss in viability. To determine the extent to which icecrystal formation was responsible for the decrease in viability postcryopreservation
TABLE 4 Viability of Hepatocytes during a 37°C Incubation Postcryopreservation Incubation medium
Time (min)
KH
0 5 10 20 30
IO0 5726 5125 34 f 5 26 -t 4
0 5 10 20 30
100 65k5 52 f 6 4024 33 + 3
LA
Fresh cells
A. In Me,SO loo 100 55k6 6o-t8 55k4 57?8 32 f 3 41 i 6 24 k 2 35 f 5
loo 6025 52 f 5 40*4 37 + 3
100 95a1 93 f 1 W&2 89 f 3
B. In 100 62+6 53 f 6 35k5 34 f 4
100 7226 69 + 8 54+5 46 + 5
100 %-+I 93 + 2 9121 89 + 3
KA
L15
25% VS 100 8OklI 66 zk 10 46+6 40 + 6
Note. Cells were incubated for varying times in Krebs-Hepes buffer (KH), KH containing 1% (w/v) bovine serum albumin (KA), Leibowitz L15 medium (L15), and Ll5 containing I% (w/v) bovine serum albumin (LA), after a 24-hr cryopreservation period at - 80°C in Me,SO cryoprotectant (A) and 25% VS (B). Values for freshly isolated cells incubated in LA medium are also shown. The values are means + SEM of between three and five experiments. Cell viability is expressed as a percentage of the viability at time 0.
(both before and after 37°C incubations), cells were cooled at a rate of f”C/min to - 10 and - 20°C (above and below the point of ice-crystal formation in the two cryoprotectants). After 15 min and 24 hr storage at these temperatures the cells were thawed rapidly and their viability was measured by trypan blue exclusion (Table 5). They were then incubated for 30 min at 37°C in Leibowitz L15 medium containing 1% (w/v) bovine serum albumin, measuring the viability of the cells at timed intervals (Table 5). The decrease in cell viabihty was less in cells cryopreserved at - 10°C than in cells cryopreserved at -20°C for 15 min. After 24 hr this effect was reversed. However, the decline in cell viability during the 37°C incubations was similar for all three storage temperatures. This would suggest that as well as ice-crystal formation, osmotic and temperature shocks may also contribute to
CRYOPRESERVATION
295
OF RAT HEPATOCYTES
TABLE 5 Postcryopreservation Cell Viability Using Different Freezing Schedules Postcryopreservation cell viability During a 37°C incubation
Freezing schedule
n
Cryoprotectant
At 4°C
5 min
10 min
20 min
30 min
i
4
ii
2
Me,SO VS25 Me,SO vs25 Me,SO VS25 Me,SO VS25 Me,SO vs25
27 + 1 41 + 1 45 55 21 32 28 44 25 39
75 2 3 79 + 5 72 86 76 90 68 71 81 82
68t4 72 k 6 61 67 68 62 64 68 73 67
62 f 5 63 2 2 58 53 39 50 46 46 37 57
38 k 7 47 k 3* 38 48 40 44 38 40 37 42
...
ill
1
iv
2
v
2
Note-The freezing schedules used were (i) 1Wmin to - 8O”C, stored for 24 hr; (ii) l*C/min to - lO”C, stored for 15 min; (iii) 1Wmin to - lO”C, stored for 24 hr; (iv) l”C/min to -2O”C, stored for 15 min; and (v) l”C/min to - 20°C stored for 24 hr. The results are means (where n B Z), with the number of experiments in parentheses. Values at 4°C refer to cell viability immediately after thawing. Cell viability at 4°C is expressed as a percentage of the fresh cell value, and for the 37°C incubation, as a percentage of the viability at time 0. * P < 0.05, when Me,SO medium is compared with VS25 medium by nonpaired Student’s t test.
the loss in cell viability during cryopreservation. To determine whether the rapid loss of viability during 37°C incubations was due to oxidation of essential cofactors or to protein degradation, 5 miI4 dithiothreitol (an antioxidant), 0.25 mM leupeptin (protease inhibitor), and 10 m&I ammonium chloride (inhibitor of lysosomal protease activity) were added individually and in combination to the washing buffers and the incubation mixtures, but no improvement in the maintenance of cell viability was observed (results not shown). Frim and Mazur (5) have suggested that the rapid loss in the viability of granulocytes incubated at 37°C postcryopreservation is due to the high lysosomal content of the cells, but this does not appear to be the case for hepatocytes. Hepatocytes were examined by transmission electron microscopy and Fig. 4 shows the typical appearance of the majority of the hepatocytes when freshly isolated (A); after 24 hr cryopreservation at -80°C followed by thawing and maintenance on ice (B), and after 30 min incubation of postcryopreserved (24 hr at -8O’C) cells at 37°C
(C). Table 6 categorizes the types of cell damage typically observed in the cells and the proportion of “normal” hepatocytes present in a sample of 100 cells examined in Figs. 4A-C. In the fresh cell samples, the majority of the cells, 65%, are normal. However, postcryopreservation, at 4°C only 28% of the ceils appeared normal, 21% showed major internal damage, and 32% showed additional partial loss of plasmalemma. After 37°C incubation, there were no structurally normal cells; most showed severe damage with partial or complete loss of plasmalemma (44 and 24% of the cells, respectively). The electron microscopic appearance of the cells postcryopreservation at 4°C was better than that described by Fuller et al. (7), who found only 10% normal cells after freezing to -60°C. DISCUSSION
For the cryopreservation of rat hepatocytes, the optimum composition of the Me,SO cryoprotectant was found to be 20% (v/v) Me,SO and 25% (v/v) FCS in Leibowitz L15 medium and for the VS, 25% (v/v) of the vitrification solution in Leibowitz L15
296
COUNDOURIS
ET AL.
0A
/ ” N
. f’
-
. .
CRYOPRESERVATION
OF RAT HEPATOCYTES
297
FIG. 4. Electron micrographs showing (A) the normal appearance of freshly isolated hepatocytes; (B) cytoplasmic swelling with lucency of organelles and glycogen clumping after 24 hr cryopreservation at -SOT, kept on ice; and (C) gross cytoplasmic and nuclear damage with plasmalemmal loss postcryopreservation and after a 37°C incubation for 30 min. Bar = 5 Frn.
medium. Freezing at a rate of l”C/min down to -80°C was the optimum freezing schedule for both cryoprotectant media. As previously observed by Fuller et al. (8) viability after thawing was lowest when the cells were plunged straight into liquid nitrogen, i.e., cooled rapidly to - 196°C directly from 4°C (20&400°C/min). Rat hepatocytes were cryopreserved for up to 7 days after freezing at 1”Clmin down to -80°C. During this period the cytochrome P450 content of the cells and the P450-dependent N-demethylation of aminopyrine were maintained at almost 100%. In fact, the N-demethylation activity of postcryopreserved cells was higher than that obtained for fresh cells (see Fig. 3), suggesting that a specific subpopulation of he-
patocytes with higher MFO activity may have survived the cryopreservation process. Previous workers have also found that the cytochrome P450-dependent metabolism of aldrin and 7-ethoxycoumarin in postcryopreserved hepatocytes is greater than in freshly isolated cells (25). Freezing at a rate of 1”Clmin down to - 80°C also resulted in improved maintenance of cytochrome P450 compared with a much more complex freezing schedule to - 196°C in a medium containing 10% Me,SO (22). In contrast to the activities of the microsomal MFO and epoxide hydrolase enzymes, glutathione-S-transferase and glutathione reductase activities fell sharply after cryopreservation. Glutathione-S-transferase activity declined to 78 f 6% of fresh
298
COUNDOURIS TABLE
6
Postcryopreservation cells (%) Cell Type
Fresh cells m
At 4°C
After 30 min at 37°C
1 2 3 4 5
65 9 17 4 4
28 4 21 32 15
0 22 10 44 24
Debris
k
+
++
Note. Rat hepatocytes, freshly isolated and after 24 hr cryopreservation in the Me,SO cryoprotectant at -WC, were examined by electron microscopy. Cryopreserved cells were either kept on ice (4°C) or incubated at 37°C in Krebs-Henseleit buffer containing 1% (w/v) bovine serum albumin for 30 min. The cells were then classified into five types depending on their morphology: (1) normal; (2) blebs on cell surface and/or clumped glycogen; (3) as 2 but with increased lucency of cytosol and organelles, dilated endoplastnic reticulum + cell swelling; (4) as 3 but more cell swelling and partial loss of plasmalemma; (5) as 4 but with total loss of plasmalemma although nucleus and cytosol still “aggregated”, the mitochondria appear swollen. Debris = isolated cell structures not in obvious “aggregates.”
cell values after 24 hr storage at - 80°C and to 34 + 7% after 7 days. The glutathione reductase activity was only 29% of fresh cell activity after 24 hr and remained at this low level throughout the 7 days. These results suggest a differential stability of the microsomal and cytosolic enzymes of hepatocytes after cryopreservation. Cytosolic enzymes may be more exposed to icecrystal damage, whereas the microsomal membrane may offer some protection to the enzymes embedded in it. These results are supported by a recent study by Powis et al. (22), where sulfotransferase activity was lost to an appreciably greater extent than UDP-glucuronosyl transferase activity in cryopreserved rat hepatocytes with biphenyl as substrate. A substantial fall in cell viability was observed after 24 hr cryopreservation at - 80°C for both cryoprotectant media used,
ET AL.
this fall being more marked for the Me,SO cryoprotectant. This initial rapid fall in viability was followed by a further slow decrease in viability over the 7-day cryopreservation period, suggesting that the actual freezing-thawing process causes greater damage to hepatocytes than does the length of the cryopreservation storage period. The gradual fall in viability over 7 days is greater in VS25 cryoprotectant, and this suggests that the vitrification solution may be more toxic to the cells. Although no significant improvement in viability was noted when the cells were stored in liquid nitrogen, it is possible that a slower freezing rate to -80°C and subsequent storage in liquid nitrogen may be beneficial for longer-term storage. A sharp fall in viability was observed during 30-min incubations at 37°C of the hepatocytes postcryopreservation; this may be partly responsible for the low MFO activities reported after cryopreservation (22), if incubation periods in excess of 10 min are used. The loss of viability is unlikely to be due to oxidative stress, as the fall in intraceilular GSH paralleled, rather than preceded, cell death. Cells cryopreserved in 25% (v/v) VS and incubated at 37°C in Leibowitz LI5 medium containing 1% (w/v) BSA showed the best maintenance of viability during the incubation (44% viable after 30 min, taking the cell viability at time 0 as 100%). This could be due to the synthesis of GSH in Leibowitz L15 medium, although Fuller et al. (8) have reported that protein synthesis is only 25% of control values, after thawing of cells frozen down to - 196°C. Karlberg and LindahlKiessling (15), describing only two samples, found no marked loss of viability in cells incubated for 1 hr at 37°C in RPM1 1640 containing 1% bovine serum albumin, after storage in liquid nitrogen. The 37°C incubation was carried out at a lower cell density, which could be responsible for the improved viability. The problem of the rapid loss in postcry-
CRYOPRESERVATION
OF RAT HEPATOCYTES
opreservation cell viability at 37°C in suspension along with the sharp decline in the activities of cytosolic enzymes during cryopreservation will have to be overcome before cryopreserved hepatocytes can be used as a model for studying drug metabolism and toxicity. However, we have promising initial results to indicate that cryopreserved rat and human adult hepatocytes attach and survive in primary culture without the rapid loss in viability observed in suspensions of cells at 37°C. The attachment of cells to the collagen-coated culture dishes may help to stabilize the cell membranes. The survival of an atypical population of hepatocytes with higher MFO activities than normal will continue to be a problem. ACKNOWLEDGMENTS We acknowledge the support of the Humane Research Trust for a Ph.D. studentship (J.A.C.), the Wellcome Trust (M.H.G.), the Scottish Home and Health Department, and the Grampian Health Board (for materials). REFERENCES 1. Carlberg, I., and Manner& B. Glutathione reductase. In “Methods in Enzymology” (A. Meister, Ed.), Vol. 113, pp. 484-490. Academic Press, New York, 1987. 2. Dansette, P. M., DuBois, G. C., and Jerina, D. M. Continuous tluorometric assay of epoxide hydrase activity. Anal. Biochem. 97,34& 345 (19791. 3. Dansette, P. M., and Jerina, D. M. A facile synthesis of arene oxides at the K regions of polycyclic hydrocarbons. J. Amer. Chem. Sot. 96, 122&1225 (1974). 4. Fahy, G. M., McFarlane, D. R., Angell, C. A., and Meryman, H. T. Vitrification as an approach to cryopreservation. Cryobidogy 21, 407426 (1984). 5. Frim, J., and Mazur, P. Interactions of cooling rate, warming rate, glycerol concentration and dilution procedure on the viability of frozenthawed human granulocytes. Cryobiology 20, 657-676 (1983). 6. Fry, J. R., and Bridges, J. W. Use of primary hepatocyte cultures in biochemical toxicology. In “Reviews in Biochemical Toxicology” (E. Hodgson, J. R. Bend, and R. M. Philpot, Eds.), Vol. 1, pp. 201-247. Elsevier/North-Holland, Amsterdam, 1979.
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7. Fuller, B. J., Grout, B. W., and Woods, R. J. Biochemical and ultrastructural examination of cryopreserved hepatocytes in rats. Cryobiology 19,493-502 (1982). 8. Fuller, B. J., Morris, G. J., Nutt, L. H., and Attenburrow, V. D. Functional recovery of isolated rat hepatocytes upon thawing from - l%“C. Cryu-Letters 1, 139-146 (1980). 9. Grant, M. H., Burke, M. D., Hawksworth, G. M., Duthie, S. J., Engeset, J., and Petrie, J. C. Human adult hepatocytes in primary monolayer culture: Maintenance of mixed function oxidase and conjugation pathways of drug metabolism. Biochem. Pharmacof. 36, 231l2316 (1987). 10. Grant, M. H., Melvin, M. A. L., Shaw, P., Melvin, W. T., and Burke, M. D. Studies on the maintenance of cytochromes P-450 and bg, monooxygenase and cytochrome reductase in primary cultures of rat hepatocytes. FEES Lett. 190, 99 (1985). 11. Guillouzo, A., Guguen-Guillouzo, C., and Bourel, M. Hepatocytes in culture: Expression of differentiated functions and their application in the study of metabolism. Triangle 20, 121-128 (1981). 12. Habig, W. H., and Jakoby, W. B. Assays for differentiation of glutaihione-S-transferase. In “Methods in Enzymology” (W. B. Jakoby, Ed.), Vol. 77, pp. 298-405. Academic Press, New York, 1981. 13. Holme, J. A., Soderlund, E., andDybing, E. Drug metabolism activities of isolated rat hepatocytes in monolayer culture. Acfa Pharmacol. Toxicol. 52, 348-356 (1983). 14. Kapitulnik, J., Wislocki, P. G., Levin, W., Yagi, H., Jerina, D. M., and Conney, A. H. Tumorigenicity studies with dial-epoxides of benzo(a)-pyrene which indicate that (+)-trans-Q,8cc, lOa-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene is an ultimate carcinogen in newborn mice. Cancer Res. 38, 354-358 (1978). 15. Karlberg, I., and Lindahl-Kiessling, K. Preservation of freshly isolated liver cells in liquid nitrogen at - 196°C.Mutat. Res. 85,411416 (1981). 16. Le Cam, A., Guillouzo, A., and Freychet, P. UItrastructural and biochemical studies of isolated adult rat hepatocytes prepared under hypoxic conditions. Cryopreservation of hepatocytes. Exp. Cell Res. 98, 382-395 (1976). 17. Levin, W., Thakker, D. R., Wood, A. W., Chang, R. L., Lebr, R. E., Jerina, D. M., and Conney, A. H. Evidence that benzo(a)anthracene 3,4diol-1,2-epoxide is an ultimate carcinogen on mouse skin. Cancer Rex. 38, 1705-1710 (1978). IS. Moldeus, P., Hogberg, J., and Orrenius, S. Isolation and use of liver cells. In “Methods in En-
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23. Reed, D. J. Regulation of reductive processes by glutathione. Eiochem. Pharmacol. 35, 7-13 (1986). 24. Saville, B. A scheme for the calorimetric determination of microgram amounts of thiols. Analyst 83, 670-672 (1958). 25. Seddon, T., Boobis, A. R., and Davies, D. S. Drug metabolising activity of cryopreserved rat and human hepatocytes. &it. J. Clin. Pharmacd. 20, 546P (1985). 26. Strom, S. C., Jirtle, R. L., Jones, R. S., Novicki, D. L., Rosenberg, M. R., Novotny, A., Irons, G., McLain, J. R., and Michalopoulos, G. Isolation, culture and transplantation of human hepatocytes. .I. NatI. Cancer Inst. 68, 771-778 (1982). 27. Walker, C. H. Species differences in microsomal monooxygenase activity and their relationship to biological half-lives. Drug Metab. Rev. 7, 295-323 (1978).
