Materials and Methods

Materials.

¹⁴C-Dextrancarboxy (6.5 mCi/ml), DMSO, polyvinyl-pyrrolidone, BSA (35%), trypsin inhibitor, L15 medium Leibovitz, and Krebs-Henseleit buffer were obtained from Sigma-Aldrich (St. Louis, MO). Tauro[carbonyl-¹⁴C]cholic acid (sodium salt) 54.0 mCi/mmol and Percoll were purchased from Amersham Pharmacia Biotech Inc. (Baie d'Urfe, QC). Collagenase type 2 was procured from Worthington Biochemical Corp. (Freehold, NJ). [³H]Estrone sulfate (specific activity, 40 Ci/mmol) and [³H]estradiol 17β-d-glucuronide (specific activity 40.5 Ci/mmol) were obtained from PerkinElmer Life Science Inc. (Boston, MA).n-Butyl phthalate was obtained from Fisher Scientific (Nepean, ON). Perchloric acid (70%) was purchased at A&C Inc. (Montreal, QC). All Falcon sterile cell culture labwares were purchased from Becton Dickinson (Franklin Lakes, NJ).

Isolation of Rat Hepatocytes.

All animal studies were approved by the institution's animal care and use committee and were conducted in accordance with all applicable regulation. Rat hepatocytes were isolated from male Sprague-Dawley rats purchased from Charles River (200–250 g) by a two-step collagenase (70 U/ml) perfusion as described by Moldéus et al. (1978). Viability of freshly isolated hepatocytes was assessed by trypan blue uptake (0.2%). Hepatocyte preparations with a cell viability <85% were rejected.

Cryopreservation and Thawing.

Cryopreservation of rat hepatocytes was conducted by two methods. The first was one described previously (Silva et al., 1999) and involved a two-step freezing protocol. The second method employed a controlled freezing protocol with a programmable freezer (Cryomed system from Thermo Forma, Marietta, OH). In these methods, freshly isolated hepatocytes were incubated for 30 min in Krebs-Henseleit buffer containing 15 mM glucose and 12 mM HEPES, pH 7.4, at 5 × 10⁶ cells/ml in a shaker-water bath at 37°C under an atmosphere of 95% O₂/5% CO₂. Cells were centrifuged and resuspended in cold L15-media containing 40% fetal bovine serum and 5% BSA, pH 7.4. Then a mixture containing an equal volume of cold L15-media containing 26% DMSO and 4% polyvinyl-pyrrolidone was added slowly (1 ml/min). Cells were transferred into 5-ml cryogenic tubes (Corning, Palo Alto, CA) and kept on ice for 10 min. For the freezing method of Method 1, the cells were placed at −20°C for 1 h and at −70°C for another hour before being stored in liquid nitrogen (Silva et al., 1999). For Method 2, we furthered assessed whether a controlled freezing procedure would improve over the two-stage freezing protocol of Method 1. A freezing procedure controlled by a Cryomed Programmable freezer (Thermo Forma) was used. That included a slow freezing at −1°C/min until the temperature reached −7°C in the sample, which is 2°C before the expected freezing point. That was followed by a supercooling step at −60°C/min to −80°C in the chamber to rapidly adsorb the release of latent heat. Then the chamber was reheated at +40°C/min to −20°C, and the freezing process was continued at −1°C/min to −40°C in the chamber. A rapid freezing step at −10°C/min to −90°C in the chamber was done to complete the freezing Method 2. Cryotubes were then rapidly placed into a liquid nitrogen storage reservoir and stored for at least 1 week prior to thawing.

When needed, hepatocytes were rapidly thawed by immersing the cryogenic tubes in a 45°C water bath just long enough to melt the solution. Iced-cold L15-media containing 0.2% BSA was added slowly 1 to 2 ml/min to dilute the DMSO, followed with washing of the cells by gentle centrifugation at 50g. The cell pellet was then resuspended in Krebs-Henseleit buffer containing 12 mM Hepes, pH 7.4, and cell viability was assessed by the Trypan blue exclusion test. A 30% isotonic Percoll centrifugation step was performed to remove dead cell when viability was lower than 85%.

Metabolism Studies.

Freshly prepared or thawed, isolated rat hepatocytes (2 × 10⁶cells/ml) were preincubated at 37°C under a 95% O₂/5% CO₂ atmosphere for 15 min before the addition of substrate. 7-Hydroxy-4-(trifluoromethyl)-coumarin (7-HFC, 100 μM) and (3,4-difluorobenzyloxy)-5,5-dimethyl-4-(4-methylsulfonylphenyl)-(5H)-furan-2-one (DFB, 250 μM), probes for UGT and CYP3A activity (Chauret et al., 1999), respectively, were incubated with the cells for 10 min. Bufuralol and tolbutamide (100 μM), probes for CYP2D and 2C (Kronbach et al., 1987; Miners et al., 1988), respectively, were incubated with the cells for 2 h. Each incubation was stopped by addition of an equal volume of acetonitrile for protein precipitation. Parent compounds remaining in the incubation mixtures and metabolites were analyzed with a high-performance liquid chromatography/UV system equipped with a Waters 717 plus auto-sampler, a Waters 616 pump, a Waters 600S controller, and a Waters 996 photodiode array detector. The data were collected and processed by Millenium version 3.20 software (Waters, Milford, MA).

The analysis of all samples was performed using a Zorbax RX-C₁₈ column, 4.6 × 150 mm, and the following detection wavelengths were used: 335 nm (7-HFC), 305 nm (DFB), 250 nm (bufuralol) and 235 nm (tolbutamide). A gradient mobile phase was used, consisting initially of 85:15, 20 mM ammonium acetate in water and acetonitrile, and was brought to 50:50 in 15 min and then to 10:90 in 3 min at a flow rate of 1.0 ml/min. Under these conditions, parent compounds (7-HFC, DFB, bufuralol, and tolbutamide) eluted at 12.7, 17.4, 12.0, and 7.5 min, respectively. The main metabolites formed were glucuronide of 7-hydroxy-4-(trifluoromethyl)-coumarin, (hydroxy)-4-(4-methyl-sulfonylphenyl)-5,5-dimethyl-(5H), 1′-OH-bufuralol, and 3-OH-tolbutamide, and eluted at 9.2, 10.8, 4.9, and 9.5 min for 7-HFC, DFB, bufuralol, and tolbutamide, respectively.

Transport Studies.

Hepatocytes suspended in Krebs-Henseleit buffer at 2 × 10⁶ cells/ml were preincubated in a shaker-water bath at 37°C for 30 min under an atmosphere of 95% O₂/5% CO₂. The uptake studies were initiated by the addition of radiolabeled substrate (1 μM final concentration) to the incubation mixture (1.4 ml final volume); sampling (125 μl) was performed in duplicates at specified times. The cells were rapidly separated from the incubation medium by a centrifugation method described by Fariss et al., 1985. Briefly, each cell aliquot was added into an Eppendorf microtube consisting of dibutyl phthalate (400 μl) layered over 10% perchloric acid (250 μl) and was centrifuged (14,000 rpm) for 15 s. The top layer was aspirated and a sample of the perchloric acid layer (150 μl) was removed into a scintillation vial. Subsequent to the addition of 15 ml of liquid scintillation fluor (Ready Protein; Beckman Canada, Mississauga, ON), the radioactivity was determined using a Beckman LS5000CE counter. The volume of adherent, extracellular fluid that was centrifuged with the cells was determined by incubating the cells with [¹⁴C]dextran-carboxy (6.5 μCi/ml), and the associated radioactivity of substrate was subtracted from the sample incubations. Data are expressed in nanomoles of compound per million of cells, after conversion of the disintegrations per minute into nanomoles from the specific activity of the sample.

Calculation and Statistics.

The initial velocity was assessed by the linear portion of the plot of the amount accumulated into cells versus time. This occurred within 1 min of the uptake study. Hence, the data points (20, 40, and 60 s) were regressed to provide the initial uptake velocity. All data were presented as the mean ± S.E.M. A Student's t test was used to test for significance.

Results

Comparison of Methods 1 and 2.

When the hepatocytes were precooled to 4°C and placed at −20°C for 1 h (Method 1), the release of latent heat occurred when the temperature in the cell mixture reached approximately −9°C with an increase of temperature of approximately 2°C, as shown in Fig.1. It took 5 min for the freezing chamber to absorb the released heat. In contrast, under conditions in which the rate of freezing was controlled and the cells were supercooled prior to the freezing point (Method 2), the release of the latent heat was dramatically minimized (Fig. 1). These conditions were found to be the ones that resulted in the least increase in temperature during the freezing process.

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Figure 1

Recording of cryopreservation.

Schematic of freezing records of cryopreservation by Method 1 (two steps, −20°C and −70°C) and Method 2 (Thermo Forma automated freezer) as described under Materials and Methodsshowing temperatures in the freezing chamber and in the cryovial as a function of time. The critical zones correspond to freezing point (−9°C), latent heat released and removed period.

Both methods for cryopreservation resulted in an initial high yield of cells with good viability as measured by trypan blue exclusion (Table1). Upon further separation of viable cells from damaged ones by Percoll centrifugation, viabilities of the resulting cells increased albeit the yields decreased. This is to be expected since the Percoll centrifugation step will remove damaged cells (less dense than intact cells), but some viable cells will also be lost. Cells cryopreserved by Method 2 consistently gave a higher yield of viable cells after Percoll centrifugation compared with cells cryopreserved by Method 1 (Table 1). Furthermore, viability of the thawed hepatocytes after a 2-h incubation was not significantly different from that of freshly isolated cells incubated over the same time periods (Table 2). In contrast, thawed cells from cryopreservation with Method 1 had significantly lower viability at the end of the 2-h incubation period (Table 2). This has also been our experience with hepatocytes obtained from other species, including dog and human (results not shown). Since we were trying to obtain the highest yield of cells after thawing, we decided at this stage to favor the Method 2 over the Method 1 to characterize cryopreserved hepatocytes in transport studies.

Metabolic Studies.

To insure that cryopreserved hepatocytes retained their metabolic capacity, substrates known to be metabolized by several cyotchromes P450 (bufuralol, tolbutamide and DFB and UGT (7-HFC) were incubated with hepatocytes before and after cryopreservation. The parent remaining after a 2-h incubation period with each probe was determined in fresh hepatocytes and compared with that observed in cryopreserved cells. Although not shown, the metabolite profiles of the probes in fresh and cryopreserved cells were identical. As shown in Fig.2, there was no significant difference in the relative metabolism of most of these probes when incubated with fresh versus thawed cryopreserved hepatocytes. Metabolism of 7-HFC was significantly decreased (p < 0.05) in cryopreserved hepatocyte compare to freshly isolated cells. About 50% of the UGT activity was lost following cryopreservation. Reports by other groups have also demonstrated that hepatocytes lose a part of conjugative pathways after cryopreservation (Diener et al., 1995; Madan et al., 1999; Steinberg et al., 1999).

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Figure 2

Metabolic activities.

Relative amount of metabolism of 7-HFC, DFB, bufuralol, and tolbutamide in freshly isolated hepatocytes (open) were compared with that observed in in cryopreserved (gray) cells as described under Materials and Methods. Isolated rat hepatocytes were cryopreserved using Method 2 as described under Materials and Methods. Data are expressed as mean ± S.E.M. ∗, significantly different from freshly prepared cells, P < 0.05 according to thet test, n = 4.

Transport Studies.

Upon the admixture of the substrate and cells to result in 1 μM of substrate (radiolabeled and unlabeled) in 1.4 ml of 2 × 10⁶ cells, aliquots (125 μl) were quickly removed, and the intracellular concentration determined by a centrifugation technique. As shown in Fig.3A, uptake of [³H]taurocholate into freshly isolated hepatocytes was rapid and linear within the first minute. The rate of accumulation was indistinguishable from that obtained with the cryopreserved hepatocytes obtained upon thawing (Fig. 3A; Table3). Similarly, the initial rates of uptake for [³H]estrone sulfate and [³H]estradiol 17β-d-glucuronide were also not significantly different in fresh cells versus the thawed cells (Fig. 3, B and C; Table 3). This limited set of results clearly suggests, for the first time, that cryopreserved hepatocytes retain their capacity to transport compounds across the plasma membrane. Furthermore, the transport of these compounds was temperature dependent and dose-saturating (results not shown), consistent with the mechanism of an active–transport mechanism. As an example, theK_m andV_max of taurocholate uptake were 36 μM and 12.3 nmol/10⁶ cells·min in freshly isolated cells, and 42 μM and 11.3 nmol/10⁶ cells·min in thawed cryopreserved (Method 2) cells.

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Figure 3

Effect of cryopreserving rat hepatocytes on the uptake transport of tracer concentrations of model compounds.

Cryopreserved or freshly isolated rat hepatocytes in Krebs-Henseleit buffer, pH 7.4, were incubated as described under Materials and Methods. [¹⁴C]taurocholate (A), [³H]estrone sulfate (B), and [³H]estradiol 17β-d-glucuronide (C) transport into hepatocytes was measured as described under Materials and Methods. Cells were incubated with 1 μM of compounds. At each time point an aliquot was spun through oil to pellet viable cells as described underMaterials and Methods. Data are expressed as mean ± S.E.M. There is no significantly difference. n = 4 (A), n = 3 (B) and (C).

Discussion

The isolated hepatocyte system that contains intact membranes serves as an important, physiologically relevant experimental tool for uptake studies. Some work has been carried out with human hepatocytes (Olinga et al., 1998a,b) whereas rat hepatocytes are used more routinely. The preparation is facile (Moldéus et al., 1978) and the intact cells contain the entire complement of transporters, including passive permeation and transporter-assisted uptake. The incubation of drug with freshly isolated suspended hepatocytes is usually conducted over a short time-frame (usually 1 min) such that the net uptake rate is mostly influx with little efflux and is time-independent. Conventional cultured cells tend to rapidly lose hepatic transport activity (Liang et al., 1993). Recently, it has been reported that hepatocytes cultured in collagen-sandwich environment regain polarity and form bile canalicular networks (Liu et al., 1998). In this model the billiary efflux transporters such as MRP2 are maintained, however, uptake transporters such as NTCP are significantly down-regulated (Liu et al., 1999). In contrast, freshly isolated hepatocytes are reported to reflect the relative uptake rates found in vivo (Sandker et al., 1994; Zhou et al., 1994). Thus, cryopreservation poses as a viable alternate procedure for the storage of human and animal hepatocytes. Cryopreserved hepatocytes have been shown to retain most of the drug-metabolizing activities (Li et al., 1999) as well as inducibility of the drug-metabolizing enzymes (Silva et al., 1999; Hengstler et al., 2000). However, use of cyropreserved hepatocytes for uptake studies has not been attempted.

We have previously described a technique for cryopreserving rat and human hepatocytes (Silva et al., 1999). Our previous, two-step freezing method (Method 1) demonstrated the importance of allowing freshly isolated hepatocytes to recover their ATP levels by preincubating the cells for 30 min at 37°C prior to freezing (Silva et al., 1999). The present programmed freezing protocol (Method 2) showed improved recovery (Table 1) and viability (Table 2). Moreover, similar phase I metabolic activities were observed (Fig. 2). Although UGT lost 50% activity, phase II metabolic activity was still observed in cryopreserved rat hepatocytes (Fig. 2).

More importantly, we demonstrated, for the first time that cryopreserved hepatocytes retained their ability in drug transport. To assess the ability of thawed cryopreserved hepatocytes to transport compounds, we focused on three well known substrates of uptake transporters and compared their uptake in thawed cryopreserved hepatocytes to freshly prepared hepatocytes that are shown to retain functional uptake transporter activities (Kato et al., 1999). Moreover, uptake by freshly prepared hepatocytes appears to be predictive of drug uptake in vivo in the liver (Akhteruzzaman et al., 1999; Kato et al., 1999; Abu-Zahra et al., 2000). As a starting point for investigation, the compounds chosen, taurocholate, estrone sulfate, and estradiol 17β-d-glucuronide, are prototypic substrates of NTCP and members of the OATP family (Hagenbuch and Meier, 1994; Jacquemin et al., 1994; Eckhardt et al., 1999; Cattori et al., 2001; Sugiyama et al., 2002). No difference in initial transport rate was found (Table3). However, the transporter functions of the organic anion transporter and organic cations by organic cationic transporter and those at the canalicular membrane, namely, P-glycoprotein and bile salt export pump, within cryopreserved hepatocytes remain unknown, although it is recognized that upon isolation, internalization of MRP2 occurs (Roelofsen et al., 1995).

In conclusion, this study demonstrates optimization in the cryopreservation of hepatocytes with Method 2. Even though cryopreservation still resulted in loss of cells, the majority of cells that survived the freezing and thawing processes appeared metabolically similar, with the exception of UGT, to those of the fresh cells. Furthermore, we also showed for the first time that cryopreserved rat hepatocytes retain drug transport activity after thawing. These preliminary observations rendered confidence for use of the cryopreserved cells in drug metabolism and transport studies. We are currently assessing this methodology to hepatocytes from other species, including human.