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Cryopreservation of precision-cut rat liver slices using a computer-controlled freezer
Toxicology in Vitro 14 (2000) 523±530
www.elsevier.com/locate/toxinvit
Cryopreservation of precision-cut rat liver slices using a computer-controlled freezer W. J. M. MAAS *, W. R. LEEMAN, J. P. GROTEN and J. J. M. VAN DE SANDT TNO Nutrition and Food Research, Department of Explanatory Toxicology, PO Box 360, 3700 AJ Zeist, The Netherlands (Accepted 25 May 2000) AbstractÐPrecision-cut liver slices are frequently used to study hepatic toxicity and metabolism of xenobiotics in vitro. Successful cryopreservation techniques will enhance an ecient and economic use of scarcely available (human) liver tissue. For primary hepatocytes, slow freezing has been accepted as the best approach towards successful cryopreservation. For slices, however, no agreement exists on the optimal way of cryopreservation and both slow and fast freezing techniques have been reported. The aim of the present study was to determine the applicability of a computer-controlled slow freezing technique for the cryopreservation of (rat) liver slices. Thus far, this technique has not been described in detail. Our studies con®rmed that slow freezing was most successful in the cryopreservation of primary rat hepatocytes. Based on this observation, the slow freezing technique was applied to the cryopreservation of rat liver slices. Directly after thawing, slice viability was between 60 and 100% of fresh values, depending on the parameter determined. However, after additional culturing, slice viability was reduced. This decrease in slice viability was more pronounced in comparison to primary hepatocytes. In conclusion, the slow freezing technique was con®rmed to be a successful approach for the cryopreservation of primary rat hepatocytes, and was found to be of limited use for the cryopreservation of rat liver slices. # 2000 Elsevier Science Ltd. All rights reserved Keywords: cryopreservation; computer-controlled freezing; rat liver slices. Abbreviations: CDNB=1-chloro-2,4-dinitrobenzene; DMSO=dimethyl sulfoxide; DNPSG=S-(2,4-dinitrophenyl)glutathione; FCS=fetal calf serum; PBS=phosphate buered saline; UW=University of Wisconsin solution; WME=Williams' medium E.
INTRODUCTION
Primary hepatocytes and precision-cut liver slices (Krumdieck et al., 1980) are used extensively to study hepatic toxicity and metabolism of xenobiotics in vitro. One of the applications of these in vitro systems in toxicological research is to gain knowledge about species dierences for selecting the most appropriate animal species to serve as a model for man. Direct application of human tissue in this type of research could make extrapolation of in vitro data to the in vivo situation more accurate. For this type of research, however, human liver tissue is only scarcely and irregularly available. Therefore, successful
*Corresponding author. Tel: +31 30 6944498; fax +31 30 6960264; e-mail: [email protected]
cryopreservation techniques for both liver slices and hepatocytes can contribute to a more economic and ecient use of tissue. A tissue bank, consisting of well characterized tissue stored for longer periods of time, would be advantageous for this purpose (Bach et al., 1996). An important issue for successful freezing protocols is the prevention of intracellular ice formation, which is considered to be the main reason for cellular damage. Intracellular ice formation might be prevented by freezing the tissue suciently slowly to allow cells to lose water in response to an increase in solute concentration in the extracellular medium. In this way, cells become dehydrated with no or minimal intracellular ice crystal formation (Mazur, 1984). On the other hand, intracellular ice formation may be avoided when cells are frozen very quickly in the presence of high concentrations of cryoprotectant,
0887-2333/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0887-2333(00)00042-4
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leading to vitri®cation of the tissue. High concentrations of cryoprotectant, however, are potentially toxic and the cooling rates needed for complete vitri®cation to occur might not be achievable in multicellular tissue (Wolfe and Bryant, 1992). From a theoretical point of view, both slow as fast freezing techniques could be considered for successful cryopreservation of liver cells and slices. For cryopreservation of primary hepatocytes, there is general consensus that (controlled) slow freezing will be the best approach towards successful cryopreservation (Li et al., 1999), and this technique is therefore applied to the freezing of liver parenchymal cells from several species (Diener et al., 1994; Powis et al., 1987; Salmon and Kohl, 1996; Swales et al., 1996). For the cryopreservation of liver slices, however, no consistent data exist. For the cryopreservation of human liver slices, for example, both slow and fast freezing techniques have been reported (Day et al., 1999; de Kanter et al., 1998; Fisher et al., 1993). Overall, both slow and fast freezing techniques have been applied to the cryopreservation of liver slices from several species using various protocols, dierent parameters to determine slice viability and with variable results (de Kanter and Koster, 1995; de Kanter et al., 1998; Ekins, 1996; Ekins et al., 1996; Fisher et al., 1991, 1993; Wishnies et al., 1991). In our laboratory, computer-controlled slow freezing is routinely used for cryopreserving single cell suspensions. This technique allows good and reproducible control of the freezing process, including the possibility of preventing the temperature rise due to crystallization. The aim of the present study was to determine the applicability of the controlled slow freezing technique to the cryopreservation of multicellular (rat) liver slices. After an initial evaluation of the slow freezing method using primary rat hepatocytes, this technique was applied to rat liver slices and the potential use of cryopreserved rat liver slices was assessed by determining a number of cell viability parameters.
carrier) and cryotubes were obtained from Costar (Cambridge, MA, USA). Collagenase type B was obtained from Boehringer Mannheim. The University of Wisconsin solution (UW) was purchased from Lamepro b.v. (Raamsdonksveer, The Netherlands). The ATP assay kit was obtained from Calbiochem-Novabiochem Corporation, (La Jolla, CA, USA). MTT was from Aldrich-Chemie (Steinheim, Germany). Testosterone and hydroxylated metabolites were from Steraloids, Inc. (Wilton, NH, USA). Isolation and culturing of primary hepatocytes Hepatocytes were isolated according to Berry and Friend (1969) and Seglen (1973) with minor modi®cations. In short, the liver was perfused with 10 mm HEPES buer (25 ml/min.) followed by a 10-min perfusion with collagenase buer (collagenase type B, 50 mg/100 ml). Hepatocytes were resuspended in WME containing 10% FCS. Hepatocytes were cultured on a roller-platform in 10 ml cryotubes under carbogen atmosphere at 37 C. Isolation and culturing of precision-cut rat liver slices Male Wistar rats were sacri®ced by decapitation and the liver was quickly removed and placed on ice in cold WME containing 10% FCS (washing medium). Slices of 200±250 mm thickness were prepared in WME from 8 mm diameter biopsies at 2±10 C using a Krumdieck tissue slicer. Slices were collected in 50-ml tubes, washed carefully in washing medium to remove debris and stored on ice until freezing or culturing (approx. 1 hr). Culture was performed in six-well plates, with Netwell inserts (two slices/well) in a humidi®ed incubator at 37 C, 40% O2/5% CO2, as previously described (Leeman et al., 1995; Olinga et al., 1997). Culture medium consisted of WME containing 0.1 mm insulin, 5% FCS, 50 mg/ml gentamicin and 25 mm d-glucose. To determine slice thickness, series of slices were cut at dierent settings of the Krumdieck tissue slicer. Wet weight was correlated to slice thickness, that was determined morphometrically. Wet weight of the slices was used to set slice thickness in further studies. Computer controlled slow freezing
MATERIALS AND METHODS
Materials Williams' medium E (WME) supplemented with Glutamax I, gentamicin and phosphate buered saline (PBS) were obtained from Gibco BRL (Paisley, Scotland, UK). Fetal calf serum (FCS) was purchased from PAA Laboratories GmbH (Linz, Austria). Insulin (from bovine pancreas), dimethyl sulfoxide (DMSO), trypan blue and 1-chloro-2,4dinitrobenzene (CDNB) were obtained from Sigma Chemical Company (St Louis, MO, USA) and dglucose from Merck (Darmstadt, Germany). Multiwell plates, Netwell inserts (200 mm polyester mesh
A SyLab Icecube 1610, Computer Freezer was used for cryopreservation of both hepatocytes and slices. Using a computer-controlled freezer, relatively large temperature rise in the sample vial, due to crystallization, can be prevented by lowering the temperature of the chamber. Primary rat hepatocytes In summary, 5 106 viable cells/ml were either slowly frozen in cryovials at 1 C/min in UW containing 10% DMSO (gradual addition of cryoprotectant on ice) or fast-frozen in UW with 10% DMSO by direct immersing the vials into liquid nitrogen. The slow freezing protocol for hepatocytes was adapted from the method described by Diener et
Slow freezing of rat liver slices
al. (1993). After slow freezing to ÿ80 C, vials were immersed in liquid nitrogen and stored in liquid nitrogen until thawing (storage time was between 1 day and 1 wk). Thawing was performed rapidly by placing the cryovials in a water-bath at 37 C under gently shaking until the ice had just melted (which took about 2 min). The medium containing the cells was then diluted 1:10 in washing medium (37 C) to remove DMSO and replaced once with fresh washing medium. Precision-cut rat liver slices The freezing protocol for liver slices was derived from the protocol used for primary hepatocytes and based on preliminary optimization experiments. Slices for cryopreservation were transferred to ice-cold UW solution containing 5% DMSO and placed on ice for approximately 10 min. Slices were then transferred to cryovials (®ve slices/vial) containing 0.5 ml UW/10% DMSO and slowly frozen as illustrated in Fig. 1. Total preincubation time with DMSO was approximately 30 min at 0±2 C, both for hepatocytes and slices. Thawing and washing of the slices was performed as described for primary hepatocytes. Viability parameters To determine urea synthesis, MTT conversion, testosterone and CDNB metabolism, slices were incubated in 12-well plates on a gyratory shaker (Gallenkamp Shaker Platform) in a humidi®ed incubator at 37 C, 40% O2/5% CO2 at 80 rpm. Urea synthesis Slices were incubated in 2 ml WME containing 10 mm NH4Cl and 1 mm ornithine. At 0, 60 and 120 min, 50-ml samples of the incubation medium were collected. Urea was determined spectrophotometrically at 540 nm using the Blood Urea Nitrogen assay kit (Sigma Chemical Company, St Louis, MO, USA). MTT conversion Slices were incubated in 2 ml WME with 0.5 mg/ ml MTT. After 1 hr, slices were removed from the medium, washed in PBS (37 C) and formazan was extracted with 1 ml DMSO in the dark overnight.
Fig. 1. Slow freeze protocol for precision-cut rat liver slices.
Formazan production was photometrically at 540 nm.
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measured
spectro-
Histomorphology Slices were ®xed in 4% buered formalin and imbedded in paran. Cross sections of 5 mm were stained with heamatoxylin and eosin. Viability was judged based on nuclear morphology and cytoplasmic staining. Metabolism of testosterone Slices were incubated in 2 ml WME containing 250 mm testosterone for 1 h. Slice and medium were stored in 2 ml cryovials at ÿ80 C until analysis. After thawing, samples were placed on ice and slices were homogenated by sonication. Hydroxylated metabolites of testosterone were extracted from 1-ml samples in two steps using dichloromethane and separated on HPLC (Hypersil ODS column, 3 200 mm, Chrompack, The Netherlands) as described (van't Klooster et al., 1993). Metabolism of CDNB Slices were incubated with 25 mm CDNB (2 ml/ well) for 15 min. Following incubation, 50 ml 0.04 m N-acetyl-l-cysteine was added to remove unreacted CDNB. Slice and medium were stored in 2 ml cryovials atÿ80 C until analysis. After thawing, samples were placed on ice and homogenated by sonication. To 200 ml homogenate, 50 ml 25% TCA was added, stored on ice for 10 min and centrifuged at 15,000 g for 5 min. S-(2,4-dinitrophenyl)glutathione (DNPSG) was quanti®ed in the supernatant by HPLC (van Iersel et al., 1996) using a ¯ow rate of 1 ml/min. and a linear gradient from 30±90% B in 20 min. A Zorbax ODS column (4.6 250 mm, Chrompack, The Netherlands) was used. Glutathione S-transferase (GST) activity and glutathione (GSH) levels Slices were blotted dry, transferred to 500 ml PBS, quickly frozen in liquid nitrogen and stored at ÿ20 C. After thawing, slices were homogenated by sonication. To 50 ml of the homogenate, 25 ml 5% (HPO3)n was added on ice. After 10 min, 225 ml 0.1 m sodium phosphate buer with 5 mm EDTA was added, vortexed and centrifuged for 5 min at 15,000 g. GSH was determined ¯uorimetrically (Shimadzu RF1501 spectro¯uorimeter; excitation wavelenght 350 nm, emission wavelength 420 nm) after reacting with o-phthalaldehyde (OPT) at pH 8.0 (Hissin and Hilf, 1976). In the same homogenate, GST activity was determined spectrophotometrically using 1 mm CDNB and 1 mm GSH as substrates. DNPSG formation was measured at 25 C for 3 min on a Varian Cary IE spectrophotometer (340 nm). Lactate dehydrogenase (LDH) activity LDH activity in the medium and the slice homogenate was measured with the BM/Hitachi 911 using
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a commercially available kit (Boehringer, Mannheim, Germany). ATP levels ATP was determined according to Singh et al. (1996) using a luciferin/luciferase assay. Slices were washed in PBS, collected in 500 ml 70% ethanol containing 2 mm EDTA, pH 10.9, quickly frozen in liquid nitrogen and stored at ÿ20 C until measurement. For ATP determination, slices were sonicated and the homogenate was centrifuged (5 min, 15,000 g). ATP was determined in the supernatant after diluting the sample 10 times with 25 mm HEPES buer, pH 7.75. To 100 ml of the diluted sample, 50 ml of the luciferin/luciferase solution was added. After mixing, light production was measured using a Lumac Biocounter M500. The pellet was resuspended in 500 ml PBS for total protein determination. Total protein Total protein was determined according to the method of Lowry et al. (1951) using bovine serum albumin (BSA) as a standard. Statistics Data are presented as mean SD. Data from fresh and cryopreserved slices (either directly after thawing of after additional culturing) were statistically evaluated using the Student's t-test. P values less than 0.05 were considered statistically signi®cant.
RESULTS
Cryopreservation of primary hepatocytes Initially, the applicability of the slow freezing technique to the cryopreservation of primary hepatocytes was evaluated. The results are summarized in Table 1. Directly after thawing it was observed that, based on trypan blue exclusion, LDH leakage and urea synthesis, about 60% of the cells were viable after slow freezing compared to approximately 20% after fast freezing. Based on the same endpoints, viability was approximately 40% and 10% for slow
and fast freezing, respectively, after 3 hr of culture. Cellular attachment was ®vefold higher in hepatocytes that were slowly frozen compared with fast frozen cells. MTT conversion in slow and fast frozen cells showed no signi®cant dierence directly after thawing. However, after 3 hr of culture, MTT conversion in fast frozen cells was approximately 50% lower compared with slow frozen cells. Cryopreservation of liver slices In preliminary experiments, several cryoprotectants and combinations of cryoprotectants (DMSO, ethylene glycol, 1,2-propanediol, 2,3-butanediol) as well as cryopreservation media (UW, WME and FCS) have been studied. The use of the UW solution as cryopreservation medium together with DMSO as a cryoprotectant gave best results using the MTT test to determine slice viability. No marked dierence in viability was observed when the liver was perfused on isolation (see Fig. 2A). No major dierences in viability were found with dierent preincubation times prior to the start of freezing. A preincubation period from 20 to 30 min was found to be optimal (see Fig. 2B). In addition, three freezing rates were tested: 0.5, 1 and 4 C/min. No signi®cant dierence was found between freezing at 0.5 or at 1 C/min, both directly after thawing or after 2 hr of additional culturing. Freezing at a rate of 4 C/min. led to a clear decrease in MTT conversion (see Fig. 2C). For further studies, a freezing rate of 0.5 C/min was used to allow optimal time for cellular dehydration to occur. The usefulness of cryopreserved rat liver slices was determined by measuring various viability parameters. Histomorphology Histomorphological examination of freshly isolated slices showed viable hepatocytes, with some damaged cells at the cutting edges of the slices. After 2 hr of culturing fresh slices, few condensed nuclei were observed together with a limited increase in eosinophilic staining. Directly after cryopreservation, histomorphology of the slices resembled the fresh situation, with a small increase in condensed nuclei and eosinophilic staining of the cytoplasm. After 2 hr in culture, dierences between freshly isolated and
Table 1. Viability of freshly isolated and cryopreserved primary rat hepatocytes
Freshly isolated Slow frozen Fast frozen
Culture time (hr)
Trypan blue exclusiona (% viable cells)
LDH leakage (% of total LDH)
0 3 0 3 0 3
885 581 565 442 183 123
n.d. 391 412 649 735 883
Urea synthesisb (% of fresh) 100
Cellular attachmentc (%) 655
513
4213
182
83
n.d.=not determined. a The cell suspension was diluted 1:1 with trypan blue (0.4%). b 3-hr incubation, percentage of urea synthesis in freshly isolated cells; 32.0 1.0 mg/106 cells/hr. c 3-hr incubation, percentage of total number of cells plated. d Data are means of two experiments (individual data are given between parentheses).
MTT conversiond (% of fresh)
96 58 48 48 26
100 (95, 97) (41, 75) (46, 50) (38, 58) (25, 27)
Slow freezing of rat liver slices
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Metabolism of testosterone The main hydroxylated metabolites formed after incubation of rat liver slices with testosterone were 6b-, 2a-, and 16a- and to a lesser extent 7a-hydroxy testosterone (-OHT) indicative for CYP3A1/2, CYP2C11, CYP2C and CYP2A, respectively. In freshly isolated slices, the major metabolites 6b- and 2a-OHT were formed to about equal extent (35 8 and 22 9 pmol/min/mg protein, respectively). The eect of cryopreservation on 6b-OHT and 2a-OHT formation is shown in Fig. 3B. Urea synthesis After cryopreservation, total urea formation was slightly, but not signi®cantly, increased from 20 to approximately 24 mg urea/mg protein during a 2-hr incubation period (data not shown). ATP and GSH levels ATP levels in freshly isolated slices increased approximately 75% during the ®rst 2 hr in culture from 3.4 to 6.0 nmol ATP/mg protein. After cryopreservation, ATP levels were 42% of fresh values and did not increase after further culturing as was observed in freshly isolated slices (see Fig. 3C). GSH levels in freshly isolated slices also increased after 2 hr in culture from 4.4 to 5.0 nmol/mg protein. Following cryopreservation, levels were about 30% lower and did not increase with time (see Fig. 3D).
Fig. 2. Preliminary studies using the MTT assay as a parameter for slice viability after slow-freezing. Preliminary studies have been performed as single experiments. Bars represents the mean of three slices SD. (A) Viability of cryopreserved rat liver slices obtained from perfused and non-perfused rat livers. Dierent cryopreservation media have been used; =FCS/10% DMSO; =WME/10% DMSO/10% FCS; =UW/10% DMSO; and =FCS/5% DMSO/4.1% ethylene glycol/10% polyvinylpyrrolidone. (*)=Statistically signi®cant, P<0.05. (B) Viability of cryopreserved rat liver slices after various preincubation periods with 10% DMSO. =directly after thawing; =after 2 hr in culture. (C) Viability of cryopreserved rat liver slices, frozen at dierent freezing rates in UW with 10% DMSO. =directly after thawing; =after 2 hr in culture.
cryopreserved slices were very marked, with almost all cells having pyknotic nuclei and the cytoplasm showing increased eosinophilic staining and loss of structure. LDH leakage Cryopreservation of liver slices clearly increased leakage of LDH during the ®rst hour of culturing compared to fresh tissue. After 2 hr of culturing cryopreserved slices, about 80% of the cells were damaged based on LDH leakage (see Fig. 3A).
GST activity GST activity was determined both in intact slices and in homogenated slices. GST activity in homogenated slices remained constant in freshly isolated slices over a culture period of 2 hr. Directly following cryopreservation, activity was 75% compared with initial values. After 2 hr of culture, GST activity was further reduced to 34% compared with fresh tissue (see Fig. 3E). When intact freshly isolated slices were incubated with CDNB, DNPSG formation increased from 0.31 to 0.40 nmol DNPSG/mg protein/min after 2 hr in culture. GST activity was reduced to 50 and 10% of initial values directly after thawing and after 2 hr in culture, respectively (see Fig. 3F), correlating with the decreased GSH levels in the slices. MTT conversion Formazan production in freshly isolated slices was slightly increased after 2 hr in culture. Following cryopreservation and 2 hr of culturing, activity was approximately 90% of that in freshly isolated tissue (see Fig. 3G).
DISCUSSION
Several attempts have been described to cryopreserve primary hepatocytes and liver slices. With respect to cryopreservation of primary isolated hepatocytes, slow freezing rates have been shown to
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Fig. 3. Viability of freshly isolated and cryopreserved rat liver slices. Bars represent the mean of three experiments SD. (*)=Statistically signi®cant, P<0.05. =fresh slices; =cryopreserved slices. (A): LDH leakage (% of total LDH), (B): metabolism of testosterone (% of freshly isolated, non-cultured slices), (C): ATP levels, (D): GSH levels, (E): GST activity in slice homogenate, (F): GST activity in intact slice, (G): MTT conversion (mean of two experiments).
yield higher amounts of viable cells compared to fast freezing rates (Chesne and Guillouzo, 1988; Diener et al., 1993; Loretz et al., 1989). In fact, there is general agreement that slow freezing is the best way to cryopreserve primary hepatocytes (Li et al., 1999), and our present results with rat hepatocytes con®rm these ®ndings. However, the optimal method for freezing multicellular liver slices has not yet been resolved, and several studies have been reported using either slow or fast freezing techniques (de Kanter and Koster, 1995; de Kanter et al., 1998; Ekins, 1996; Ekins et al., 1996; Fisher et al., 1991, 1993; GloÈckner et al., 1998; Wishnies et al., 1991).
In this study we investigated the applicability of a computer-controlled slow freezing technique to the preservation of multicellular rat liver slices. This technique allows reproducible controlled-rate freezing with the possibility to adjust for the temperature rise at crystallization. Furthermore, this method avoids the use of high cryoprotectant concentrations. Using this technique, slice viability directly after thawing, was comparable to freshly isolated tissue based on urea synthesis and MTT reduction. An increase in the leakage of LDH and the histomorphological examination, however, indicated that some damage to cellular membranes was apparent.
Slow freezing of rat liver slices
The reduced GST activity in the slice homogenate as well is most likely to be caused by enzyme leakage due to membrane damage. After 2 hr in culture, viability of cryopreserved liver tissue declined as could be concluded from most viability endpoints. This loss of viability was more pronounced in comparison to primary hepatocytes. MTT conversion in slices, however, remained relatively constant and did not correlate well with the other parameters determined. These results are not in line with those obtained by McGann et al. (1988), showing that the MTT assay is a sensitive test to detect freeze-thaw damage in cells. A possible reason for the rapid loss of tissue viability shortly after thawing is that dehydration of cells imbedded in the original architecture in the slice has not been sucient (Bishof et al., 1997) to prevent for intracellular ice crystals to form. Furthermore, cell volume changes as a result of introduction and removal of the cryoprotectant, and exposure of membranes (and cellular proteins) to high electrolyte concentrations due to cellular dehydration during slow freezing, may cause irreversible damage (Levin and Miller, 1981). In addition, while hepatocytes may be able to adapt in size during slow freezing, cells imbedded in their original matrix might be less able to adapt in shape during the dehydration process (due to intact cell-cell contacts), leading more easily to cell membrane damage. Another reason for the loss of cell integrity might be the insucient cryoprotectant penetration into the tissue. It has been demonstrated that equilibration of rat liver cores (2 mm thickness) with DMSO takes a relatively long time (Fuller et al., 1994). However, since slices are far less thick, they can be expected to be almost completely equilibrated with the surrounding medium. From the present study it can be concluded that computer-controlled freezing of rat liver slices has only limited success. Other studies do also indicate that rat liver slices rapidly lose viability following slow freezing and subsequent short-term culturing (de Kanter and Koster, 1995; Fisher et al., 1991). In these studies, however, no attempt was described to prevent temperature rise due to ice nucleation, although it is generally agreed on that, at least for primary hepatocytes, this is an important feature for successful cryopreservation (Li et al., 1999). Interestingly, slow freezing of liver slices from species other than rat (pig and human), was reported to be quite successful up to 4 hr after additional culture, although results with human tissue showed large variation that seemed to be dependent on the length of warm and cold ischemia prior to the freezing procedure (Fisher et al., 1991, 1993). Species dierences in resistance to slow freezing have also been mentioned for hepatocytes, in which primary cells from rat liver were more susceptible to freezing stress compared to the cells obtained from several other species (Diener et al., 1993; Salmon and
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Kohl, 1996). Our results therefore agree with the published literature, suggesting that rat liver tissue is highly susceptible to slow freezing stress. In conclusion, for the freezing of primary hepatocytes, controlled slow freezing was con®rmed to be the optimal approach for successful cryopreservation. Directly after thawing, viability of multicellular rat liver slices was found to be close to fresh values, whereas a pronounced decrease in tissue viability was observed after additional short-term culturing. We therefore believe that at present the main applicability of cryopreserved rat liver slices is in short-term metabolism studies. Further studies now focus on species dierences in sensitivity towards cryopreservation and on the assessment of critical parameters in the fast and slow freezing process. AcknowledgementsÐPart of this work was ®nanced by Biotech, Bio4 CT 972145 and the TNO Network on Alternatives to Animal Testing. REFERENCES
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Olinga P., Groen K., Hof I. H., de Kanter R., Koster H. J., Leeman W. R., Rutten A. A. J. J. L., van Twillert K. and Groothuis G. M. M. (1997) Comparison of ®ve incubation systems for rat liver slices using functional and viability parameters. Journal of Pharmacological and Toxicological Methods 38, 59±69. Powis G., Santone K. S., Melder K. C., Thomas L., Moore D. J. and Wilke T. J. (1987) Cryopreservation of rat and dog hepatocytes for studies of xenobiotic metabolism and activation. Drug Metabolism and Disposition 15, 826±832. Salmon F. and Kohl W. (1996) Use of fresh and cryopreserved hepatocytes to study the metabolism of pesticides in food-producing animals and rats. Xenobiotica 26, 803± 811. Seglen P. O. (1973) Preparation of rat liver cells III. Enzymatic requirements for tissue dispersion. Experimental Cell Research 82, 391±398. Singh Y., Cooke J. B., Hinton D. E. and Miller M. G. (1996) Trout liver slices for metabolism and toxicity studies. Drug Metabolism and Disposition 24, 7±14. Swales N. J., Johnson T. and Caldwell J. (1996) Cryopreservation of rat and mouse hepatocytes II. Assessment of metabolic capacity using testosterone metabolism. Drug Metabolism and Disposition 24, 1224±1230. van Iersel M. L. P. S., Ploemen J.-P. H. T. M., Struik I., van Amersfoort C., Keyzer A. E., Scheerlie J. G. and van Bladeren P. J. (1996) Inhibition of glutathione S-transferase activity in human melanoma cells by -unsaturated carbonyl derivates. Eects of acrolein, cinnamaldehyde, citral, crotonaldehyde, curcumin, ethacrynic acid, and trans-2-hexenal. Chemico-Biological Interactions 102, 117±132. van't Klooster G. A. E., Blaauboer B. J., Noordhoek J. and van Miert A. S. J. P. A. M. (1993) Cytochrome P450 induction and metabolism of alkoxyresoru®ns, ethylmorphine and testosterone in cultured hepatocytes from goats, sheep and cattle. Biochemical Pharmacology 46, 1781±1790. Wishnies S. M., Parrish A. R., Sipes I. G., Gandol® A. J., Putnam C. W., Krumdieck C. L. and Brendel K. (1991) Biotransformation activity in vitri®ed human liver slices. Cryobiology 28, 216±226. Wolfe J. and Bryant G. (1992). Physical principles of membrane damage due to dehydration and freezing. In Mechanics of Swelling, ed. T. K. Karalis, Vol. H 64, pp. 205±224. Springer-Verlag Berlin.
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Cryopreservation of precision-cut rat liver slices using a computer-controlled freezer W. J. M. MAAS *, W. R. LEEMAN, J. P. GROTEN and J. J. M. VAN DE SANDT TNO Nutrition and Food Research, Department of Explanatory Toxicology, PO Box 360, 3700 AJ Zeist, The Netherlands (Accepted 25 May 2000) AbstractÐPrecision-cut liver slices are frequently used to study hepatic toxicity and metabolism of xenobiotics in vitro. Successful cryopreservation techniques will enhance an ecient and economic use of scarcely available (human) liver tissue. For primary hepatocytes, slow freezing has been accepted as the best approach towards successful cryopreservation. For slices, however, no agreement exists on the optimal way of cryopreservation and both slow and fast freezing techniques have been reported. The aim of the present study was to determine the applicability of a computer-controlled slow freezing technique for the cryopreservation of (rat) liver slices. Thus far, this technique has not been described in detail. Our studies con®rmed that slow freezing was most successful in the cryopreservation of primary rat hepatocytes. Based on this observation, the slow freezing technique was applied to the cryopreservation of rat liver slices. Directly after thawing, slice viability was between 60 and 100% of fresh values, depending on the parameter determined. However, after additional culturing, slice viability was reduced. This decrease in slice viability was more pronounced in comparison to primary hepatocytes. In conclusion, the slow freezing technique was con®rmed to be a successful approach for the cryopreservation of primary rat hepatocytes, and was found to be of limited use for the cryopreservation of rat liver slices. # 2000 Elsevier Science Ltd. All rights reserved Keywords: cryopreservation; computer-controlled freezing; rat liver slices. Abbreviations: CDNB=1-chloro-2,4-dinitrobenzene; DMSO=dimethyl sulfoxide; DNPSG=S-(2,4-dinitrophenyl)glutathione; FCS=fetal calf serum; PBS=phosphate buered saline; UW=University of Wisconsin solution; WME=Williams' medium E.
INTRODUCTION
Primary hepatocytes and precision-cut liver slices (Krumdieck et al., 1980) are used extensively to study hepatic toxicity and metabolism of xenobiotics in vitro. One of the applications of these in vitro systems in toxicological research is to gain knowledge about species dierences for selecting the most appropriate animal species to serve as a model for man. Direct application of human tissue in this type of research could make extrapolation of in vitro data to the in vivo situation more accurate. For this type of research, however, human liver tissue is only scarcely and irregularly available. Therefore, successful
*Corresponding author. Tel: +31 30 6944498; fax +31 30 6960264; e-mail: [email protected]
cryopreservation techniques for both liver slices and hepatocytes can contribute to a more economic and ecient use of tissue. A tissue bank, consisting of well characterized tissue stored for longer periods of time, would be advantageous for this purpose (Bach et al., 1996). An important issue for successful freezing protocols is the prevention of intracellular ice formation, which is considered to be the main reason for cellular damage. Intracellular ice formation might be prevented by freezing the tissue suciently slowly to allow cells to lose water in response to an increase in solute concentration in the extracellular medium. In this way, cells become dehydrated with no or minimal intracellular ice crystal formation (Mazur, 1984). On the other hand, intracellular ice formation may be avoided when cells are frozen very quickly in the presence of high concentrations of cryoprotectant,
0887-2333/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0887-2333(00)00042-4
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leading to vitri®cation of the tissue. High concentrations of cryoprotectant, however, are potentially toxic and the cooling rates needed for complete vitri®cation to occur might not be achievable in multicellular tissue (Wolfe and Bryant, 1992). From a theoretical point of view, both slow as fast freezing techniques could be considered for successful cryopreservation of liver cells and slices. For cryopreservation of primary hepatocytes, there is general consensus that (controlled) slow freezing will be the best approach towards successful cryopreservation (Li et al., 1999), and this technique is therefore applied to the freezing of liver parenchymal cells from several species (Diener et al., 1994; Powis et al., 1987; Salmon and Kohl, 1996; Swales et al., 1996). For the cryopreservation of liver slices, however, no consistent data exist. For the cryopreservation of human liver slices, for example, both slow and fast freezing techniques have been reported (Day et al., 1999; de Kanter et al., 1998; Fisher et al., 1993). Overall, both slow and fast freezing techniques have been applied to the cryopreservation of liver slices from several species using various protocols, dierent parameters to determine slice viability and with variable results (de Kanter and Koster, 1995; de Kanter et al., 1998; Ekins, 1996; Ekins et al., 1996; Fisher et al., 1991, 1993; Wishnies et al., 1991). In our laboratory, computer-controlled slow freezing is routinely used for cryopreserving single cell suspensions. This technique allows good and reproducible control of the freezing process, including the possibility of preventing the temperature rise due to crystallization. The aim of the present study was to determine the applicability of the controlled slow freezing technique to the cryopreservation of multicellular (rat) liver slices. After an initial evaluation of the slow freezing method using primary rat hepatocytes, this technique was applied to rat liver slices and the potential use of cryopreserved rat liver slices was assessed by determining a number of cell viability parameters.
carrier) and cryotubes were obtained from Costar (Cambridge, MA, USA). Collagenase type B was obtained from Boehringer Mannheim. The University of Wisconsin solution (UW) was purchased from Lamepro b.v. (Raamsdonksveer, The Netherlands). The ATP assay kit was obtained from Calbiochem-Novabiochem Corporation, (La Jolla, CA, USA). MTT was from Aldrich-Chemie (Steinheim, Germany). Testosterone and hydroxylated metabolites were from Steraloids, Inc. (Wilton, NH, USA). Isolation and culturing of primary hepatocytes Hepatocytes were isolated according to Berry and Friend (1969) and Seglen (1973) with minor modi®cations. In short, the liver was perfused with 10 mm HEPES buer (25 ml/min.) followed by a 10-min perfusion with collagenase buer (collagenase type B, 50 mg/100 ml). Hepatocytes were resuspended in WME containing 10% FCS. Hepatocytes were cultured on a roller-platform in 10 ml cryotubes under carbogen atmosphere at 37 C. Isolation and culturing of precision-cut rat liver slices Male Wistar rats were sacri®ced by decapitation and the liver was quickly removed and placed on ice in cold WME containing 10% FCS (washing medium). Slices of 200±250 mm thickness were prepared in WME from 8 mm diameter biopsies at 2±10 C using a Krumdieck tissue slicer. Slices were collected in 50-ml tubes, washed carefully in washing medium to remove debris and stored on ice until freezing or culturing (approx. 1 hr). Culture was performed in six-well plates, with Netwell inserts (two slices/well) in a humidi®ed incubator at 37 C, 40% O2/5% CO2, as previously described (Leeman et al., 1995; Olinga et al., 1997). Culture medium consisted of WME containing 0.1 mm insulin, 5% FCS, 50 mg/ml gentamicin and 25 mm d-glucose. To determine slice thickness, series of slices were cut at dierent settings of the Krumdieck tissue slicer. Wet weight was correlated to slice thickness, that was determined morphometrically. Wet weight of the slices was used to set slice thickness in further studies. Computer controlled slow freezing
MATERIALS AND METHODS
Materials Williams' medium E (WME) supplemented with Glutamax I, gentamicin and phosphate buered saline (PBS) were obtained from Gibco BRL (Paisley, Scotland, UK). Fetal calf serum (FCS) was purchased from PAA Laboratories GmbH (Linz, Austria). Insulin (from bovine pancreas), dimethyl sulfoxide (DMSO), trypan blue and 1-chloro-2,4dinitrobenzene (CDNB) were obtained from Sigma Chemical Company (St Louis, MO, USA) and dglucose from Merck (Darmstadt, Germany). Multiwell plates, Netwell inserts (200 mm polyester mesh
A SyLab Icecube 1610, Computer Freezer was used for cryopreservation of both hepatocytes and slices. Using a computer-controlled freezer, relatively large temperature rise in the sample vial, due to crystallization, can be prevented by lowering the temperature of the chamber. Primary rat hepatocytes In summary, 5 106 viable cells/ml were either slowly frozen in cryovials at 1 C/min in UW containing 10% DMSO (gradual addition of cryoprotectant on ice) or fast-frozen in UW with 10% DMSO by direct immersing the vials into liquid nitrogen. The slow freezing protocol for hepatocytes was adapted from the method described by Diener et
Slow freezing of rat liver slices
al. (1993). After slow freezing to ÿ80 C, vials were immersed in liquid nitrogen and stored in liquid nitrogen until thawing (storage time was between 1 day and 1 wk). Thawing was performed rapidly by placing the cryovials in a water-bath at 37 C under gently shaking until the ice had just melted (which took about 2 min). The medium containing the cells was then diluted 1:10 in washing medium (37 C) to remove DMSO and replaced once with fresh washing medium. Precision-cut rat liver slices The freezing protocol for liver slices was derived from the protocol used for primary hepatocytes and based on preliminary optimization experiments. Slices for cryopreservation were transferred to ice-cold UW solution containing 5% DMSO and placed on ice for approximately 10 min. Slices were then transferred to cryovials (®ve slices/vial) containing 0.5 ml UW/10% DMSO and slowly frozen as illustrated in Fig. 1. Total preincubation time with DMSO was approximately 30 min at 0±2 C, both for hepatocytes and slices. Thawing and washing of the slices was performed as described for primary hepatocytes. Viability parameters To determine urea synthesis, MTT conversion, testosterone and CDNB metabolism, slices were incubated in 12-well plates on a gyratory shaker (Gallenkamp Shaker Platform) in a humidi®ed incubator at 37 C, 40% O2/5% CO2 at 80 rpm. Urea synthesis Slices were incubated in 2 ml WME containing 10 mm NH4Cl and 1 mm ornithine. At 0, 60 and 120 min, 50-ml samples of the incubation medium were collected. Urea was determined spectrophotometrically at 540 nm using the Blood Urea Nitrogen assay kit (Sigma Chemical Company, St Louis, MO, USA). MTT conversion Slices were incubated in 2 ml WME with 0.5 mg/ ml MTT. After 1 hr, slices were removed from the medium, washed in PBS (37 C) and formazan was extracted with 1 ml DMSO in the dark overnight.
Fig. 1. Slow freeze protocol for precision-cut rat liver slices.
Formazan production was photometrically at 540 nm.
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measured
spectro-
Histomorphology Slices were ®xed in 4% buered formalin and imbedded in paran. Cross sections of 5 mm were stained with heamatoxylin and eosin. Viability was judged based on nuclear morphology and cytoplasmic staining. Metabolism of testosterone Slices were incubated in 2 ml WME containing 250 mm testosterone for 1 h. Slice and medium were stored in 2 ml cryovials at ÿ80 C until analysis. After thawing, samples were placed on ice and slices were homogenated by sonication. Hydroxylated metabolites of testosterone were extracted from 1-ml samples in two steps using dichloromethane and separated on HPLC (Hypersil ODS column, 3 200 mm, Chrompack, The Netherlands) as described (van't Klooster et al., 1993). Metabolism of CDNB Slices were incubated with 25 mm CDNB (2 ml/ well) for 15 min. Following incubation, 50 ml 0.04 m N-acetyl-l-cysteine was added to remove unreacted CDNB. Slice and medium were stored in 2 ml cryovials atÿ80 C until analysis. After thawing, samples were placed on ice and homogenated by sonication. To 200 ml homogenate, 50 ml 25% TCA was added, stored on ice for 10 min and centrifuged at 15,000 g for 5 min. S-(2,4-dinitrophenyl)glutathione (DNPSG) was quanti®ed in the supernatant by HPLC (van Iersel et al., 1996) using a ¯ow rate of 1 ml/min. and a linear gradient from 30±90% B in 20 min. A Zorbax ODS column (4.6 250 mm, Chrompack, The Netherlands) was used. Glutathione S-transferase (GST) activity and glutathione (GSH) levels Slices were blotted dry, transferred to 500 ml PBS, quickly frozen in liquid nitrogen and stored at ÿ20 C. After thawing, slices were homogenated by sonication. To 50 ml of the homogenate, 25 ml 5% (HPO3)n was added on ice. After 10 min, 225 ml 0.1 m sodium phosphate buer with 5 mm EDTA was added, vortexed and centrifuged for 5 min at 15,000 g. GSH was determined ¯uorimetrically (Shimadzu RF1501 spectro¯uorimeter; excitation wavelenght 350 nm, emission wavelength 420 nm) after reacting with o-phthalaldehyde (OPT) at pH 8.0 (Hissin and Hilf, 1976). In the same homogenate, GST activity was determined spectrophotometrically using 1 mm CDNB and 1 mm GSH as substrates. DNPSG formation was measured at 25 C for 3 min on a Varian Cary IE spectrophotometer (340 nm). Lactate dehydrogenase (LDH) activity LDH activity in the medium and the slice homogenate was measured with the BM/Hitachi 911 using
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a commercially available kit (Boehringer, Mannheim, Germany). ATP levels ATP was determined according to Singh et al. (1996) using a luciferin/luciferase assay. Slices were washed in PBS, collected in 500 ml 70% ethanol containing 2 mm EDTA, pH 10.9, quickly frozen in liquid nitrogen and stored at ÿ20 C until measurement. For ATP determination, slices were sonicated and the homogenate was centrifuged (5 min, 15,000 g). ATP was determined in the supernatant after diluting the sample 10 times with 25 mm HEPES buer, pH 7.75. To 100 ml of the diluted sample, 50 ml of the luciferin/luciferase solution was added. After mixing, light production was measured using a Lumac Biocounter M500. The pellet was resuspended in 500 ml PBS for total protein determination. Total protein Total protein was determined according to the method of Lowry et al. (1951) using bovine serum albumin (BSA) as a standard. Statistics Data are presented as mean SD. Data from fresh and cryopreserved slices (either directly after thawing of after additional culturing) were statistically evaluated using the Student's t-test. P values less than 0.05 were considered statistically signi®cant.
RESULTS
Cryopreservation of primary hepatocytes Initially, the applicability of the slow freezing technique to the cryopreservation of primary hepatocytes was evaluated. The results are summarized in Table 1. Directly after thawing it was observed that, based on trypan blue exclusion, LDH leakage and urea synthesis, about 60% of the cells were viable after slow freezing compared to approximately 20% after fast freezing. Based on the same endpoints, viability was approximately 40% and 10% for slow
and fast freezing, respectively, after 3 hr of culture. Cellular attachment was ®vefold higher in hepatocytes that were slowly frozen compared with fast frozen cells. MTT conversion in slow and fast frozen cells showed no signi®cant dierence directly after thawing. However, after 3 hr of culture, MTT conversion in fast frozen cells was approximately 50% lower compared with slow frozen cells. Cryopreservation of liver slices In preliminary experiments, several cryoprotectants and combinations of cryoprotectants (DMSO, ethylene glycol, 1,2-propanediol, 2,3-butanediol) as well as cryopreservation media (UW, WME and FCS) have been studied. The use of the UW solution as cryopreservation medium together with DMSO as a cryoprotectant gave best results using the MTT test to determine slice viability. No marked dierence in viability was observed when the liver was perfused on isolation (see Fig. 2A). No major dierences in viability were found with dierent preincubation times prior to the start of freezing. A preincubation period from 20 to 30 min was found to be optimal (see Fig. 2B). In addition, three freezing rates were tested: 0.5, 1 and 4 C/min. No signi®cant dierence was found between freezing at 0.5 or at 1 C/min, both directly after thawing or after 2 hr of additional culturing. Freezing at a rate of 4 C/min. led to a clear decrease in MTT conversion (see Fig. 2C). For further studies, a freezing rate of 0.5 C/min was used to allow optimal time for cellular dehydration to occur. The usefulness of cryopreserved rat liver slices was determined by measuring various viability parameters. Histomorphology Histomorphological examination of freshly isolated slices showed viable hepatocytes, with some damaged cells at the cutting edges of the slices. After 2 hr of culturing fresh slices, few condensed nuclei were observed together with a limited increase in eosinophilic staining. Directly after cryopreservation, histomorphology of the slices resembled the fresh situation, with a small increase in condensed nuclei and eosinophilic staining of the cytoplasm. After 2 hr in culture, dierences between freshly isolated and
Table 1. Viability of freshly isolated and cryopreserved primary rat hepatocytes
Freshly isolated Slow frozen Fast frozen
Culture time (hr)
Trypan blue exclusiona (% viable cells)
LDH leakage (% of total LDH)
0 3 0 3 0 3
885 581 565 442 183 123
n.d. 391 412 649 735 883
Urea synthesisb (% of fresh) 100
Cellular attachmentc (%) 655
513
4213
182
83
n.d.=not determined. a The cell suspension was diluted 1:1 with trypan blue (0.4%). b 3-hr incubation, percentage of urea synthesis in freshly isolated cells; 32.0 1.0 mg/106 cells/hr. c 3-hr incubation, percentage of total number of cells plated. d Data are means of two experiments (individual data are given between parentheses).
MTT conversiond (% of fresh)
96 58 48 48 26
100 (95, 97) (41, 75) (46, 50) (38, 58) (25, 27)
Slow freezing of rat liver slices
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Metabolism of testosterone The main hydroxylated metabolites formed after incubation of rat liver slices with testosterone were 6b-, 2a-, and 16a- and to a lesser extent 7a-hydroxy testosterone (-OHT) indicative for CYP3A1/2, CYP2C11, CYP2C and CYP2A, respectively. In freshly isolated slices, the major metabolites 6b- and 2a-OHT were formed to about equal extent (35 8 and 22 9 pmol/min/mg protein, respectively). The eect of cryopreservation on 6b-OHT and 2a-OHT formation is shown in Fig. 3B. Urea synthesis After cryopreservation, total urea formation was slightly, but not signi®cantly, increased from 20 to approximately 24 mg urea/mg protein during a 2-hr incubation period (data not shown). ATP and GSH levels ATP levels in freshly isolated slices increased approximately 75% during the ®rst 2 hr in culture from 3.4 to 6.0 nmol ATP/mg protein. After cryopreservation, ATP levels were 42% of fresh values and did not increase after further culturing as was observed in freshly isolated slices (see Fig. 3C). GSH levels in freshly isolated slices also increased after 2 hr in culture from 4.4 to 5.0 nmol/mg protein. Following cryopreservation, levels were about 30% lower and did not increase with time (see Fig. 3D).
Fig. 2. Preliminary studies using the MTT assay as a parameter for slice viability after slow-freezing. Preliminary studies have been performed as single experiments. Bars represents the mean of three slices SD. (A) Viability of cryopreserved rat liver slices obtained from perfused and non-perfused rat livers. Dierent cryopreservation media have been used; =FCS/10% DMSO; =WME/10% DMSO/10% FCS; =UW/10% DMSO; and =FCS/5% DMSO/4.1% ethylene glycol/10% polyvinylpyrrolidone. (*)=Statistically signi®cant, P<0.05. (B) Viability of cryopreserved rat liver slices after various preincubation periods with 10% DMSO. =directly after thawing; =after 2 hr in culture. (C) Viability of cryopreserved rat liver slices, frozen at dierent freezing rates in UW with 10% DMSO. =directly after thawing; =after 2 hr in culture.
cryopreserved slices were very marked, with almost all cells having pyknotic nuclei and the cytoplasm showing increased eosinophilic staining and loss of structure. LDH leakage Cryopreservation of liver slices clearly increased leakage of LDH during the ®rst hour of culturing compared to fresh tissue. After 2 hr of culturing cryopreserved slices, about 80% of the cells were damaged based on LDH leakage (see Fig. 3A).
GST activity GST activity was determined both in intact slices and in homogenated slices. GST activity in homogenated slices remained constant in freshly isolated slices over a culture period of 2 hr. Directly following cryopreservation, activity was 75% compared with initial values. After 2 hr of culture, GST activity was further reduced to 34% compared with fresh tissue (see Fig. 3E). When intact freshly isolated slices were incubated with CDNB, DNPSG formation increased from 0.31 to 0.40 nmol DNPSG/mg protein/min after 2 hr in culture. GST activity was reduced to 50 and 10% of initial values directly after thawing and after 2 hr in culture, respectively (see Fig. 3F), correlating with the decreased GSH levels in the slices. MTT conversion Formazan production in freshly isolated slices was slightly increased after 2 hr in culture. Following cryopreservation and 2 hr of culturing, activity was approximately 90% of that in freshly isolated tissue (see Fig. 3G).
DISCUSSION
Several attempts have been described to cryopreserve primary hepatocytes and liver slices. With respect to cryopreservation of primary isolated hepatocytes, slow freezing rates have been shown to
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Fig. 3. Viability of freshly isolated and cryopreserved rat liver slices. Bars represent the mean of three experiments SD. (*)=Statistically signi®cant, P<0.05. =fresh slices; =cryopreserved slices. (A): LDH leakage (% of total LDH), (B): metabolism of testosterone (% of freshly isolated, non-cultured slices), (C): ATP levels, (D): GSH levels, (E): GST activity in slice homogenate, (F): GST activity in intact slice, (G): MTT conversion (mean of two experiments).
yield higher amounts of viable cells compared to fast freezing rates (Chesne and Guillouzo, 1988; Diener et al., 1993; Loretz et al., 1989). In fact, there is general agreement that slow freezing is the best way to cryopreserve primary hepatocytes (Li et al., 1999), and our present results with rat hepatocytes con®rm these ®ndings. However, the optimal method for freezing multicellular liver slices has not yet been resolved, and several studies have been reported using either slow or fast freezing techniques (de Kanter and Koster, 1995; de Kanter et al., 1998; Ekins, 1996; Ekins et al., 1996; Fisher et al., 1991, 1993; GloÈckner et al., 1998; Wishnies et al., 1991).
In this study we investigated the applicability of a computer-controlled slow freezing technique to the preservation of multicellular rat liver slices. This technique allows reproducible controlled-rate freezing with the possibility to adjust for the temperature rise at crystallization. Furthermore, this method avoids the use of high cryoprotectant concentrations. Using this technique, slice viability directly after thawing, was comparable to freshly isolated tissue based on urea synthesis and MTT reduction. An increase in the leakage of LDH and the histomorphological examination, however, indicated that some damage to cellular membranes was apparent.
Slow freezing of rat liver slices
The reduced GST activity in the slice homogenate as well is most likely to be caused by enzyme leakage due to membrane damage. After 2 hr in culture, viability of cryopreserved liver tissue declined as could be concluded from most viability endpoints. This loss of viability was more pronounced in comparison to primary hepatocytes. MTT conversion in slices, however, remained relatively constant and did not correlate well with the other parameters determined. These results are not in line with those obtained by McGann et al. (1988), showing that the MTT assay is a sensitive test to detect freeze-thaw damage in cells. A possible reason for the rapid loss of tissue viability shortly after thawing is that dehydration of cells imbedded in the original architecture in the slice has not been sucient (Bishof et al., 1997) to prevent for intracellular ice crystals to form. Furthermore, cell volume changes as a result of introduction and removal of the cryoprotectant, and exposure of membranes (and cellular proteins) to high electrolyte concentrations due to cellular dehydration during slow freezing, may cause irreversible damage (Levin and Miller, 1981). In addition, while hepatocytes may be able to adapt in size during slow freezing, cells imbedded in their original matrix might be less able to adapt in shape during the dehydration process (due to intact cell-cell contacts), leading more easily to cell membrane damage. Another reason for the loss of cell integrity might be the insucient cryoprotectant penetration into the tissue. It has been demonstrated that equilibration of rat liver cores (2 mm thickness) with DMSO takes a relatively long time (Fuller et al., 1994). However, since slices are far less thick, they can be expected to be almost completely equilibrated with the surrounding medium. From the present study it can be concluded that computer-controlled freezing of rat liver slices has only limited success. Other studies do also indicate that rat liver slices rapidly lose viability following slow freezing and subsequent short-term culturing (de Kanter and Koster, 1995; Fisher et al., 1991). In these studies, however, no attempt was described to prevent temperature rise due to ice nucleation, although it is generally agreed on that, at least for primary hepatocytes, this is an important feature for successful cryopreservation (Li et al., 1999). Interestingly, slow freezing of liver slices from species other than rat (pig and human), was reported to be quite successful up to 4 hr after additional culture, although results with human tissue showed large variation that seemed to be dependent on the length of warm and cold ischemia prior to the freezing procedure (Fisher et al., 1991, 1993). Species dierences in resistance to slow freezing have also been mentioned for hepatocytes, in which primary cells from rat liver were more susceptible to freezing stress compared to the cells obtained from several other species (Diener et al., 1993; Salmon and
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Kohl, 1996). Our results therefore agree with the published literature, suggesting that rat liver tissue is highly susceptible to slow freezing stress. In conclusion, for the freezing of primary hepatocytes, controlled slow freezing was con®rmed to be the optimal approach for successful cryopreservation. Directly after thawing, viability of multicellular rat liver slices was found to be close to fresh values, whereas a pronounced decrease in tissue viability was observed after additional short-term culturing. We therefore believe that at present the main applicability of cryopreserved rat liver slices is in short-term metabolism studies. Further studies now focus on species dierences in sensitivity towards cryopreservation and on the assessment of critical parameters in the fast and slow freezing process. AcknowledgementsÐPart of this work was ®nanced by Biotech, Bio4 CT 972145 and the TNO Network on Alternatives to Animal Testing. REFERENCES
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