Journal of Pharmacological and Toxicological Methods 73 (2015) 63–71

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Original article

Drug-induced cholestasis detection in cryopreserved rat hepatocytes in sandwich culture Marlies Oorts a, Lysiane Richert b,c, Pieter Annaert a,⁎ a b c

Drug Delivery and Disposition, KU Leuven, Department of Pharmaceutical and Pharmacological Sciences, O&N2, Herestraat 49, Box 921, 3000 Leuven, Belgium KaLy-Cell, 20A rue du Général Leclerc, 67115 Plobsheim, France Université de Franche-Comté, 25030 Besançon, France

a r t i c l e

i n f o

Article history: Received 24 December 2014 Received in revised form 4 March 2015 Accepted 16 March 2015 Available online 28 March 2015 Keywords: Cyclosporin A Cryopreserved hepatocytes Drug-induced cholestasis Sandwich-cultured rat hepatocytes Troglitazone

a b s t r a c t Introduction: In vitro identification of compounds that cause cholestasis in vivo still remains a problem in pharmaceutical R&D. Currently existing in vitro systems show poor predictivity towards the clinical situation. Recently, our research group developed a model, based on sandwich-cultured (rat) hepatocytes (SC(R)H), to detect compounds causing cholestasis via altered bile acid (BA) homeostasis (Chatterjee et al., 2014). In the present study, we assessed whether this model performs equally well with freshly-isolated and cryopreserved hepatocytes. Methods: We exposed sandwich cultures from rat hepatocytes before and after cryopreservation to the cholestatic compounds, cyclosporin A (CsA) and troglitazone (Tro), in the presence and in the absence of a BA mixture. At the end of the incubations, the capability of the hepatocytes to produce urea was measured to determine changes in the drug-induced cholestasis index (DICI). Results: The mean (± SEM) urea production was significantly higher in sandwich cultures from freshlyisolated hepatocytes (27.88 (± 0.96) nmol/cm2), compared to cultures from cryopreserved hepatocytes (22.86 (± 1.91) nmol urea/cm2). However, after normalization for confluence rate (based on light microscopic image analysis), it appeared that the urea production was similar for all the batches of SCRH. The mean (± SEM) DICI values for CsA 10 μM and Tro 75 μM were 0.89 (± 0.03) and 0.93 (± 0.03), respectively. Higher concentrations, CsA (≥ 15 μM) and Tro (≥ 100 μM), elicited a significant decrease in urea production when incubated in the presence of a BA mixture compared to the compound alone. This was the case for all the batches of SCRH, irrespective of cryopreservation history. Discussion: In conclusion, no significant differences were seen when the previously described in vitro cholestasis model was applied in SCRH before or after cryopreservation. This study demonstrates the robustness of the model, which implies that it can be used with SCRH obtained from both freshly-isolated and cryopreserved hepatocytes. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Abbreviations: AUC, area under the curve; BA(s), bile acid(s); Bsep/BSEP, bile salt export pump (rat/human); CDCA, chenodeoxycholic acid; CDF, 5-(6)-carboxy-2′,7′dichlorofluorescein; CDFDA, 5-(6)-carboxy-2′,7′-dichlorofluorescein diacetate; CsA, cyclosporin A; DCA, deoxycholic acid; DIC, drug-induced cholestasis; DICI, drug-induced cholestasis index; DILI, drug-induced liver injury; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethylsulfoxide; GCA, glycocholic acid; GCDCA, glycochenodeoxycholic acid; GDCA, glycodeoxycholic acid; FBS, fetal bovine serum; HBSS, Hanks' balanced salt solution; HEPES, 4-2-hydroxyethyl-1-piperazineethanesulfonic acid; LOAEL, lowest observed adverse effect level; Mrp2/MRP2, multidrug resistance associated protein 2 (rat/human); Ntcp/ NTCP, Na+-dependent taurocholate co-transporting polypeptide (rat/human); PBS, phosphate buffered saline; SCH, sandwich-cultured hepatocytes; SCHH, sandwich-cultured human hepatocytes; SCRH, sandwich-cultured rat hepatocytes; Tro, troglitazone; WEM, Williams' E medium. ⁎ Corresponding author. Tel.: +32 16 33 03 03. E-mail addresses: [email protected] (M. Oorts), [email protected], [email protected] (L. Richert), [email protected] (P. Annaert).

http://dx.doi.org/10.1016/j.vascn.2015.03.002 1056-8719/© 2015 Elsevier Inc. All rights reserved.

Drug-induced liver injury (DILI) remains a major health problem with broad implications for patient healthcare, regulatory agencies and pharmaceutical industry (Ghabril, Chalasani, & Björnsson, 2010). More than 47% of the registered DILI cases are the result of cholestatic injury, which implies that drug-induced cholestasis (DIC) accounts for one of the major mechanisms of (drug-induced) hepatotoxicity (Andrade et al., 2005; Björnsson & Olsson, 2005; Chalasani et al., 2008; De Valle, Av Klinteberg, Alem, Olsson, & Björnsson, 2006; Friis & Andreasen, 1992; Kleiner et al., 2014; Wagner, Zollner, & Trauner, 2009). DIC is caused by any interference with the physiological function of the transport proteins and enzymes related to bile acid (BA) disposition (e.g. uptake of BAs into the hepatocytes, metabolism/conjugation, denovo BA synthesis and efflux of the BAs from the hepatocytes). This disturbance will lead to an intracellular accumulation of BAs together with

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M. Oorts et al. / Journal of Pharmacological and Toxicological Methods 73 (2015) 63–71

other cholephiles in the liver, and will result in hepatocellular toxicity (Stieger, Fattinger, Madon, Kullak-Ublick, & Meier, 2000). As a consequence, compounds causing hepatotoxicity should be identified during early stages of drug development prior to clinical studies and approval (Dawson, Stahl, Paul, Barber, & Kenna, 2012). Unfortunately, currently existing in vitro and in vivo animal models for DILI offer limited predictive results (Au, Navarro, & Rossi, 2011). In particular, to investigate the cholestatic potential of a compound, several in vitro systems have been applied, such as selectively isolated basolateral and canalicular rat liver plasma membrane vesicles, bile salt export pump (Bsep/BSEP) overexpressing membrane vesicles, a porcine kidney epithelial cell line coexpressing Na+-dependent taurocholate co-transporting polypeptide (Ntcp/NTCP) and Bsep/BSEP (Dawson et al., 2012; Mita et al., 2006; Moseley, Johnson, & Morrissette, 1990; Pedersen et al., 2013). These systems mostly rely on the assessment of inhibitory capacity of a compound towards the canalicular efflux transporter(s), specifically Bsep/ BSEP, suggesting that inhibition of this transporter is denoted as the sole mechanism of DIC (Morgan et al., 2010; Stieger et al., 2000). However, as already mentioned above, recent studies suggest the role of other mechanisms underlying DIC, including interference with uptake of BAs into the hepatocytes, their metabolism/conjugation and de-novo BA synthesis (Rodrigues et al., 2014; Yang, Köck, Sedykh, Tropsha, & Brouwer, 2013). Therefore, we hypothesized that an in vitro model which mimics the impact of disturbed BA homeostasis underlying a cholestatic effect on cell health and functions is expected to better predict cholestatic potential in vivo. The utility of sandwich-cultured hepatocytes (SCH) to investigate DILI mechanisms, and DIC in particular has been shown previously (De Bruyn et al., 2013). Indeed, hepatocytes cultured in a sandwich configuration have the ability to maintain functional expression levels of both uptake and efflux transport proteins, as well as phase I and II metabolism pathways for several days (De Bruyn et al., 2013; Richert et al., 2002; Richert et al., 2009). Therefore, SCH allow studying xenobiotics that can disturb the different pathways of BA homeostasis at the hepatic level. Based on this concept, our research group developed an in vitro assay using SCH for the identification of compounds that have the possibility to cause cholestasis by altering BA homeostasis (Chatterjee, Richert, Augustijns and Annaert, 2014b). The drug-induced cholestasis index (DICI) was introduced as a new parameter to reflect the risk of cholestasis in vitro, as determined by comparing the capability of the SCH incubated with a compound of interest in the absence and in the presence of a physiologically relevant BA mixture to convert ammonia to urea. One advantage of the urea assay is that it is based on the detection of sublethal reductions in hepatocyte functionality of the hepatocytes, while other, more commonly used cytotoxicity assays, mostly rely on a ‘dead or alive principle’. Another great advantage of this assay is its nondestructive nature, which makes it possible to recycle cells after urea assessment. In the present study, we assessed whether our in vitro model for cholestasis risk assessment on sandwich-cultured rat hepatocytes (SCRH) can perform equally well when using either freshly-isolated or cryopreserved hepatocytes. Therefore, we incubated SCRH with two compounds, cyclosporin A (CsA) and trogitazone (Tro), in the presence and in the absence of a BA mixture. Subsequently, we determined the urea production obtained by SCRH established from cells before and after cryopreservation. A comparison was made between the DICI values of both compounds obtained in several batches of freshlyisolated and cryopreserved rat hepatocytes. 2. Materials and methods 2.1. Materials Williams' E Medium (WEM), Dulbecco's Modified Eagle's Medium (DMEM), L-glutamine, penicillin–streptomycin mixture (containing 10,000 IU/mL potassium penicillin and 10,000 μg/mL streptomycin

sulfate), fetal bovine serum (FBS), Hanks' balanced salt solution (HBSS; referred to as standard buffer with pH adjusted to 7.4), phosphate buffered saline (PBS; 1 × and 10 ×), and trypan blue solution (0.4%) were purchased from Lonza Westburg BV (Leusden, The Netherlands). ITS+™ Premix (contains 6.25 mg/L insulin, 6.25 mg/L transferin, 6.25 mg/L selenious acid, 1.25 g/L bovine serum albumin and 5.35 mg/L linoleic acid) was obtained from BD Biosciences (Erembodegem, Belgium). HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid) was acquired from MP Biochemical (Illkirch, France). Chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA), collagenase type IV (from Clostridium histolyticum), recombinant human insulin, dexamethasone, urea, diacetyl monoxime, thiosemicarbazide, iron (III) chloride hexahydrate, orthophosphoric acid, ornithine, DMEM 10 ×, Percoll® and 5-(6)-carboxy-2′,7′dichlorofluorescein diacetate (CDFDA) were purchased from SigmaAldrich (Diegem, Belgium). Ammonium chloride was obtained from UCB (Brussels, Belgium). Sulfuric acid (95–97%) was purchased from Chem-Lab NV (Zedelgem, Belgium). Sodium hydroxide was obtained from Merck KGaA (Darmstadt, Germany). CsA and Tro were purchased from Sequoia Research Products Ltd. (Pangbourne, UK). 24-well sterile cell culture plates were acquired from Greiner Bio-One BVBA (Wemmel, Belgium). Thermostable 96-well plates were kindly provided by Greiner Bio-One BVBA (Wemmel, Belgium). Collagen was prepared in-house from rat tails according to established procedures.

2.2. Animals Male Wistar rats (170–200 g) were housed in the Central Animal Facilities of KU Leuven, according to the guidelines and policies for animal experiments, housing and care, and the laws of Belgium and European Union. Studies were approved by the Institutional Ethical Committee for Animal Experimentation of KU Leuven. Rats were maintained in a 12 h light–dark cycle with free access to water and standard rat/mouse maintenance food (ssniff Spezialdiäten GmbH, Germany).

2.3. Hepatocyte isolation Hepatocytes were isolated from male Wistar rats based on a twostep collagenase perfusion method, as described previously (Annaert, Turncliff, Booth, Thakker, & Brouwer, 2001), without addition of trypsin inhibitor. Rats were anesthetized by an intraperitoneal injection of a mixture of xylazine (24 mg/kg) and ketamine (120 mg/kg). After isolation, cells were centrifuged (50 g) for 3 min at 4 °C and the pellet was re-suspended in WEM containing 5% FBS, 2 mM L-glutamine, 100 IU/mL penicillin, and 100 μg/mL streptomycin. Hepatocytes were counted using a hemocytometer and cell viability was determined using trypan blue. Subsequently, cells were either cultured in a sandwich configuration or cryopreserved as described below.

2.4. Cryopreservation of rat hepatocytes The cell suspension obtained after isolation was centrifuged (50 g) at 4 °C for 3 min. Supernatant was aspirated and cells were re-suspended in cryoprotective medium (Synth-a-freeze® Cryopreservation medium, Gibco®, Ghent, Belgium) at a cell density of 10 × 106 cells/mL. Cells were cryopreserved in a programmable controlled rate freezer, Kryo 560–16 (Planer, Sunbury-on-Thames, UK), during a freezing cycle containing a supercooling phase. After reaching − 100 °C, the cryovials were stored in liquid nitrogen until thawing.

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Table 1 The characteristics of the freshly-isolated and cryopreserved rat hepatocyte batches used during this study. The gray area indicates the viability, recovery and confluence after cryopreservation, thawing and culturing of the hepatocytes.

Batch n°

Viability (%)

Yield (× 106 cells/mL)

Confluence

Post–

Recovery

Confluence

(±SD) (%)

thawing viability

(%)

(±SD) (%)

(%) 1

89

6.8

82.6 (±2.9)

84

75

54.8 (±4.3)

2

87

8.0

85.0 (±2.9)

81

59

71.8 (±6.1)

3

92

10.0

93.0 (±1.8)

79

75

91.1 (±1.1)

4

90

12.5

96.7 (±4.4)

74

74

73.7 (±1.5)

5

91

11.5

93.6 (±5.4)

79

72

64.3 (±4.3)

6

96

10.6

81.5 (±0.5)

76

45

47.9 (±3.5)

7

93

11.5

93.0 (±2.7)

70

63

–

8

92

5.5

88.6 (±6.3)

9

90

9.4

73.5 (±1.9)

10

82

5.3

78.8 (±8.3)

11

93

11.0

89.2 (±3.5)

12

86

11.0

91.1 (±2.3)

2.5. Thawing of cryopreserved rat hepatocytes Cryovials were removed from liquid nitrogen and immersed into a 37 °C water bath. Immediately after all ice crystals were melted, hepatocytes were re-suspended in 16 mL Percoll® solution (90% Percoll® and 10% 10× PBS) and 24 mL thawing medium (DMEM supplemented with 10% FBS, 100 IU/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, 4 μg/mL insulin and 1 μM dexamethasone). The volume was adjusted to 50 mL with thawing medium (37 °C) and centrifuged at 168 g for 20 min at room temperature. Subsequently, supernatant was removed and the pellet was re-suspended in 20 mL thawing medium. The suspension was centrifuged a second time (50 g) for 3 min at room temperature. Before culturing the hepatocytes, the cells were resuspended in seeding medium (consisting of WEM supplemented with 10% FBS, 100 IU/mL penicillin, 100 μg/mL streptomycin, 2 mM Lglutamine, 4 μg/mL insulin and 1 μM dexamethasone). Hepatocytes were counted using a hemocytometer and cell viability was determined using trypan blue.

culturing. The characteristics of the freshly-isolated and cryopreserved rat hepatocyte batches used during this study are listed in Table 1. Hepatocytes used in experiments had a viability after isolation of at least 70%. Rat hepatocytes were cultured in sandwich configuration as previously described (Chatterjee et al., 2014b). Briefly, 24-well plates were coated with ice-cold neutralized collagen solution (~ 1.5 mg/mL, pH 7.4), placed overnight at 37 °C in a humidified incubator, and hydrated with PBS (500 μL/well) before use. Hepatocytes were seeded at a density of 0.5 × 106 cells/well. After incubating the cells at 37 °C in a humidified atmosphere with 5% CO2 (binder CO2 incubator, binder GmbH) for 1–2 h, unattached cells were removed by shaking the plate and immediately aspirating the medium. To obtain a sandwich configuration, the cells were overlaid with 50 μL of rat tail collagen solution (~1.5 mg/mL, pH 7.4). One hour later, day-0 medium (or seeding medium) (37 °C) was added onto the cultures which were kept in a humidified atmosphere with 5% CO2 (day-0). The medium was changed every day with culture medium consisting of WEM supplemented with 1% (v/v) ITS+™ Premix, 2 mM L-glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin, and 0.1 μM dexamethasone (day-1 medium).

2.6. Sandwich-cultured rat hepatocytes 2.7. Incubation with test compound and BAs After determining viability and yield of both freshly-isolated and cryopreserved rat hepatocytes, the cell suspension was further diluted with day-0 medium (WEM containing 5% FBS, 2 mM L-glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin, 4 μg/mL insulin, and 1 μM dexamethasone) or seeding medium (in case of cryopreserved hepatocytes) to obtain a final cell density of 106 cells/mL, prior to

A sixty-fold concentrated solution of a BA mixture (referred to as 60× BA mixture) consisting of the five quantitatively most important BAs present in human plasma, were used (Gnewuch, 2009; Scherer, Gnewuch, Schmitz, & Liebisch, 2009; Xiang et al., 2010). Hepatocytes were first incubated with the test compound alone for 2 h, to provide

Fig. 1. Morphology of sandwich-cultured rat hepatocytes SCRH obtained from freshly-isolated rat hepatocytes. Light microscopic images of SCRH at day-1 (D1) and day-3 (D3) of culture time, representative for all the batches used during this study (magnification: 100×).

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Fig. 2. Morphology of sandwich-cultured rat hepatocytes (SCRH) obtained from cryopreserved rat hepatocytes. Light microscopic images of SCRH at day-1 (D1) and day-3 (D3) of culture time, representative for all the batches used during this study (magnification: 100×).

the test compound time to interfere with the hepatic transporters. Subsequently, the incubation medium was replaced by a mixture of the compound (at the same concentration) and the 60 × BA mixture, and incubated for 22 h. After the incubations, a urea assay was performed for a quantitative assessment of (compromised) hepatocyte functionality. All the solutions were prepared in day-1 medium. 2.8. Determination of urea production in SCRH The capacity of the hepatocytes to convert ammonia to urea was used to assess the overall biochemical function and integrity of rat hepatocytes. Urea production by SCRH was determined as described previously (Chatterjee, Bijsmans, van Mil, Augustijns & Annaert, 2014a; Chatterjee et al., 2014b). Briefly, the cells were washed twice with standard buffer and incubated with pre-warmed (37 °C) standard buffer containing 10 mM HEPES, 2 mM glutamine, 10 mM ammonium chloride and 3 mM ornithine (250 μL/well) for 1 h at 37 °C in a humidified atmosphere with 5% CO2. Next, 60 μL of the incubation buffer/well was mixed with 240 μL of color reagent in a 96-well thermostable plate. This mixture was heated at 85 °C for 20 min in a water bath and subsequently cooled down by keeping the plate at 4 °C for 10 min. Absorbance was measured at 525 nm using a Tecan Infinite M200 plate reader (Tecan Group Ltd., Männedorf, Austria).

2.10. Data analysis To quantify the ability of a test compound to exert toxicity by disturbing BA homeostasis in vitro, a drug-induced cholestasis index (DICI) was calculated as follows (Chatterjee et al., 2014b):

DICI ¼

Urea production test compound þ BAs : Urea production test compound alone

DICI values were calculated for each compound at every concentration examined. When a compound at a particular concentration gave a DICI value ≤ 0.8, the compound was flagged positive for cholestasis in vitro. A DICI N 0.8 signified no risk for cholestasis in vitro. 2.11. Statistics

2.9. Light and fluorescence microscopic imaging

Data are represented as mean ± SD or mean ± SEM. A two-tailed paired Student's t-test in Microsoft excel (version 2010) was used to evaluate statistical significance of differences between urea production (nmol/cm2) in SCRH obtained from freshly-isolated and cryopreserved hepatocytes. Differences were considered statistically significant at *p b 0.05; **p b 0.01 and ***p b 0.001. All graphs and Area Under the Curve (AUC) ratios between urea production of the test compound in the presence of BAs and urea production of the test compound alone were made using GraphPad Prism 5.0 for Windows (California, USA).

The biliary excretory function in SCRH was assessed by qualitative evaluation of 5-(6)- carboxy-2′,7′-dichlorofluorescein (CDF) excretion in bile canalicular networks via fluorescence microscopy (ex/em 490/ 520 nm). SCRH at day-3 of culture time were washed twice with standard buffer (37 °C) and incubated with standard buffer containing 10 mM HEPES for 10 min. Next, hepatocytes were incubated with 4 μM CDFDA in standard buffer containing 10 mM HEPES for another 10 min, after which the buffer was removed. Afterwards, the hepatocytes and biliary network were imaged (both by fluorescence and light microscopy) with a VisiCam® 3.0 camera (VWR International Bvba, Leuven, Belgium), mounted on a Olympus IX70 inverted tissue culture microscope (Olympus Optical Co GMBH, Hamburg, Germany). A monochromator (Polychrome IV; Till Photonics, Oberhausen, Germany) was used to generate the excitation wavelength (490 nm). For fluorescence microscopy a U-MWIB3 mirror unit with a long pass emission filter (510 nm) and a dichroic mirror (505 nm) was used. Light microscopic images of both freshly-isolated and cryopreserved rat hepatocytes were taken daily. Confluence was determined with Image J software (NIH, version 1.48 s) by manual selection of areas covered by hepatocytes.

Fig. 3. Comparison of the mean (±SEM) urea production, in corresponding freshly-isolated and cryopreserved rat hepatocyte batches in sandwich configuration at day-4 of culture time. Each bar represents mean (±SEM) urea production obtained in 7 different batches of rat hepatocytes (n = 7). The bars on the right represent urea production after normalization for confluence (%) of the individual cultures. p b 0.05 (Student's t-test) for pairwise comparison between urea production of freshly-isolated and cryopreserved hepatocyte batches. NS: Not significant.

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3.2. Biliary excretion of CDF in SCRH SCRH were exposed to the fluorogenic probe CDFDA to assess the canalicular excretory function of the hepatocytes. CDFDA passively diffuses into the hepatocytes, where it is hydrolyzed rapidly by intracellular esterases to the fluorophore CDF. The latter is excreted into the bile canaliculi by the multidrug resistance associated protein 2 (Mrp2/ MRP2). Extensive and functional biliary networks were present in SCRH at day-3 of culture time, which is depicted in Supplemental Fig. 1. 3.3. Biochemical functionality of SCRH from corresponding batches of freshly-isolated and cryopreserved rat hepatocytes The capability of hepatocytes to convert ammonia to urea was assessed in 7 batches of rat hepatocytes before and after cryopreservation. A comparison of the mean urea production in day-4 SCRH from 7 batches of rat hepatocytes before and after cryopreservation is shown in Fig. 3. The mean (± SEM) urea production observed in SCRH from freshly-isolated hepatocytes (27.9 (±1.0) nmol/cm2) was significantly (p b 0.05) higher than the mean (±SEM) urea production observed in SCRH from cryopreserved hepatocytes (22.9 (±1.9) nmol/cm2). However, after normalization of the urea production to the confluence of each individual culture, no difference between both conditions could be observed (p = 0.20). 3.4. Effect of co-incubation with a BA mixture on the toxicity of CsA in freshly-isolated and cryopreserved batches of SCRH Different concentrations (10–20 μM) of CsA were incubated in the absence and in the presence of the 60× BA mixture in various batches (n = 10) of freshly-isolated rat hepatocytes to determine the effect of BAs on the concentration-dependent toxicity of this cholestatic compound. Fig. 4 illustrates the urea production (normalized for confluence) in SCRH from freshly-isolated (Panel A; n = 10) and two individual batches of cryopreserved rat hepatocytes (Panels B and C) at day-4 of culture time when incubated with different concentrations of CsA in the absence and in the presence of a 60 × BA mixture for 24 h. The toxicity exerted at 15 and 20 μM CsA was significantly

Fig. 4. Concentration-dependent effect of cyclosporin A (CsA) on urea production in day-4 sandwich-cultured rat hepatocytes (SCRH). Day-3 SCRH were incubated for 24 h with different concentrations of CsA in the absence (black bars) and in the presence (gray bars) of a 60 × concentrated bile acid (BA) mixture. Panel A. Values obtained in 10 batches of freshly-isolated rat hepatocytes. Each bar represents the mean (± SEM, n = 10) urea production, normalized for the confluence of each individual batch of SCRH. Panels B and C. Values obtained in 2 individual batches of cultures prepared from cryopreserved rat hepatocytes. Each bar represents mean (± SD, n = 3) urea production in SCRH. *p b 0.05, **p b 0.01 and ***p b 0.001 (Student's t-test) for pair-wise comparison between conditions in the absence (black bars) and conditions in the presence (gray bars) of the 60 × BA mixture.

3. Results 3.1. Morphology of freshly-isolated and cryopreserved rat hepatocytes in sandwich configuration The mean (± SEM) confluence of SCRH prepared from either freshly-isolated or cryopreserved rat hepatocytes was 87.2 ± 2.0 and 68.3 ± 6.2%, respectively. This difference in confluence is depicted in Figs. 1 and 2, which are representative light microscopic images of SCRH from freshly-isolated and cryopreserved hepatocytes at day-1 and day-3 of culture time. More bile canaliculi were present in SCRH at day-3 compared to day-1 of culture time.

Fig. 5. Drug-induced cholestasis index (DICI) of different concentrations of cyclosporin A (CsA) in freshly-isolated rat hepatocytes in sandwich configuration. Points represent the mean DICI values obtained in 10 batches of sandwich-cultured rat hepatocytes (SCRH). Lines represent the overall mean (±SEM) DICI values. CsA was incubated with and without a bile acid (BA) mixture, as described in the Materials and methods section, and DICI values were calculated based on the relative urea production. The dotted line on the Yaxis represents a DICI value of 0.8.

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Table 2 Drug-induced cholestasis index (DICI) values of cyclosporin A (CsA) in sandwich-cultured rat hepatocytes (SCRH) before and after cryopreservation. Mean (±SD) DICI values of different concentrations of CsA of freshly-isolated rat hepatocytes, compared to cryopreserved rat hepatocytes of the corresponding batch. Concentration

DICI (±SD) of batch #1

Cyclosporin A (μM)

Before cryopreservation

After cryopreservation

DICI (±SD) of batch #2 Before cryopreservation

After cryopreservation

10 15 20

0.74 (±0.08) 0.28 (±0.05) 0.31 (±0.06)

0.70 (±0.13) 0.49 (±0.04) 0.42 (±0.09)

0.90 (±0.08) 0.80 (±0.15) 0.50 (±0.05)

0.94 (±0.05) 0.70 (±0.07) 0.56 (±0.08)

higher in the presence of BAs in all SCRH from both freshly-isolated and cryopreserved hepatocytes. The toxicity of 10 μM CsA was also increased in the presence of BAs in freshly-isolated and one batch of cryopreserved SCRH. It should be noted that the urea production, obtained in SCRH from freshly-isolated hepatocytes, was significantly lower in the control group with BAs, compared to the control group without BAs. Nevertheless, DICI values still remained higher than 0.8. DICI values were calculated based on the capability of hepatocytes to produce urea and are depicted in Fig. 5. A concentration-dependent decrease of DICI values was observed in all batches of freshly-isolated rat hepatocytes. At 10, 15 and 20 μM of CsA the mean (±SEM) DICI values, obtained in SCRH from freshly-isolated hepatocytes were found to be 0.89 (±0.03), 0.51 (±0.04) and 0.42 (±0.03), respectively. This implicates that at 15 and 20 μM of CsA, the DICI values were found to be lower than or equal to 0.8. In Table 2, the DICI values are depicted from two individual cryopreserved rat hepatocytes batches. For 10 μM of CsA, batch #1 yielded a DICI value lower than 0.8, while this was not the case for batch #2. The AUC values (Supplemental Fig. 2) and the corresponding ratio of AUC values in the presence or in the absence of the 60× BA mixture in all freshly-isolated and cryopreserved rat hepatocyte batches (Table 3) were thereafter calculated and compared. Based on the Student's t-test, no significant differences of the ratios (with BA/without BA) were found between batches of both freshly-isolated and cryopreserved rat hepatocytes. The concentration-dependent toxicity is also reflected in the light microscopic images of all conditions tested during this study (Supplemental Fig. 4a). 3.5. Effect of co-incubation with a BA mixture on the toxicity of Tro in freshly-isolated and cryopreserved batches of SCRH Tro (50–125 μM) was incubated in the absence and in the presence of the 60× BA mixture in various batches (n = 10) of rat hepatocytes before and after cryopreservation. Tro, incubated at a concentration of 50 μM in freshly-isolated or cryopreserved hepatocytes, did not yield any decrease in urea production, even in the presence of the BA mixture, as shown in Fig. 6. In contrast, the urea production of SCRH incubated with 75, 100 and 125 μM Tro in the presence of the BA mixture was significantly decreased compared to the compound alone. Except for one batch of SCRH of cryopreserved hepatocytes, where no significant effect was observed at 75 μM Tro. DICI values, obtained from freshly-isolated hepatocytes (n = 10) and cryopreserved hepatocytes (n = 2–4) are depicted in Figs. 7 and 8, respectively. A similar concentrationdependent decrease of DICI values was observed in sandwich cultures obtained from both freshly-isolated and cryopreserved hepatocytes. More specifically, at 50, 75, 100 and 125 μM of Tro, the mean (±SEM)

DICI values obtained in SCRH from freshly-isolated hepatocytes were found to be 0.93 (± 0.03), 0.83 (± 0.04), 0.37 (± 0.03) and 0.31 (±0.03), respectively. For 125 μM of Tro, in total 3 values were excluded due to the low urea production of the SCRH of the compound alone (below the 60% cut-off for urea production). Table 4 shows the ratio of AUC values in the presence or in the absence of the 60× BA mixture in batches of freshly-isolated and cryopreserved rat hepatocytes. No significant differences in AUC ratios were found between batches of both freshly-isolated and cryopreserved rat hepatocytes. The concentrationdependent toxicity of Tro is reflected in light microscopic images of all conditions used during this study (Supplemental Fig. 4b). 4. Discussion The aim of this study was to assess the inter-changeability of freshlyisolated and cryopreserved rat hepatocytes in sandwich culture for incubations with cholestatic compounds. Therefore, we incubated SCRH with two model compounds (CsA and Tro) in the absence and in the presence of a bio-relevant BA mixture. The results obtained in the present study illustrate that both freshlyisolated and cryopreserved rat hepatocytes, cultivated in a sandwich configuration, can be used as an in vitro model to identify compounds with a potential to cause cholestasis. In order to reach this conclusion, several aspects of the experiment design, in particular the confluence of the cultures, the toxicity of the compounds alone and the incubation time, had to be taken into account. It was noticed that the urea production of the control group in SCRH at day-4 of culture time was significantly higher in cultures seeded from freshly-isolated hepatocytes compared to cultures obtained from cryopreserved hepatocytes from the same donor. However, when normalizing the urea production for the confluence rate of the cultures, no significant differences were observed. Indeed, based on the light microscopic images, SCRH obtained from freshly-isolated hepatocytes exhibit higher confluence, compared to cultures established from cryopreserved hepatocytes. This is due to the fact that the attachment after thawing rat hepatocytes was reduced, as previously reported (Grondin, Hamel, Averill-Bates, & Sarhan, 2009; Swales, Luong, & Caldwell, 1996). It has been hypothesized that structural membrane damage might contribute to lower plating efficiency (Stéphenne, Najimi, & Sokal, 2010). When CsA (10–20 μM) and Tro (50–125 μM) were incubated in SCRH, no statistically significant differences were observed between the results obtained in freshly-isolated hepatocytes and cryopreserved hepatocytes. Overall, a concentration-dependent increase in cholestatic potential was noticed, which was in concordance with previously

Table 3 Comparison of the Area under the Curve (AUC) values in the presence and in the absence of a bile acid (BA) mixture in freshly-isolated and cryopreserved rat hepatocyte batches. The concentration-dependent effect of cyclosporin A on urea production of sandwich-cultured rat hepatocytes (SCRH), as shown in Supplemental Fig. 2, was used to calculate the AUC values by means of the trapezoidal rule. Cyclosporin A

AUC (without BAs)

AUC (with BAs)

Ratio (with BAs/without BAs)

Before cryopreservation (n = 10) Batch #1; after cryopreservation Batch #2; after cryopreservation

780.9 790.7 861.2

474.6 421.4 642.1

0.61 0.53 0.75

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obtained results of Chatterjee et al. (2014b). More specifically, an enhanced toxicity was observed when the compounds were incubated in the presence of the BA mixture. This enhanced toxicity started at 10 and 75 μM for CsA and Tro, respectively. Nevertheless, both compounds at corresponding concentrations did not always yield DICI values lower than 0.8 in all the batches of rat hepatocytes used during the study. These concentrations are expected to be very close to the lowest observed adverse effect level (LOAEL) of cholestasis in vitro. Higher concentrations, namely 15 μM CsA and 100 μM Tro always yielded DICI values ≤ 0.8 in sandwich cultures of both freshly-isolated and cryopreserved hepatocytes, suggesting that these concentrations are recommended positive conditions in future experiments. Overall, matching results were obtained with paired batches before and after cryopreservation (i.e. 10 μM CsA yielded a DICI value higher than 0.8 in one batch of freshly isolated hepatocytes, but also in the corresponding culture of cryopreserved hepatocytes).

Fig. 7. Drug-induced cholestasis index (DICI) of different concentrations of troglitazone in freshly-isolated rat hepatocytes in sandwich configuration. Points represent the individual mean DICI values obtained in 10 batches of sandwich-cultured rat hepatocytes (SCRH). For 125 μM Tro, 3 values were excluded due to the low urea production of the compound alone (below the urea baseline production of 60%). Lines represent the overall average (± SEM) DICI values. Tro was incubated with and without bile acid (BA) mixture and DICI values were calculated based on the relative urea production. The dotted line on the Y-axis represents a DICI value of 0.8.

Furthermore, it was observed that the compound alone at higher concentrations (e.g. 20 μM CsA and 125 μM Tro) rendered toxicity in some batches of SCRH. This effect was slightly more pronounced in cultures of cryopreserved hepatocytes, implicating that cells after cryopreservation and thawing were slightly more sensitive towards the toxic effects. Nevertheless, this observation had a negligible impact on the corresponding DICI value. If the compounds were incubated in the presence of BAs, the toxicity increased, meaning that the BAs sensitize the hepatocytes towards the cholestatic action of the compound of interest. Importantly, if the compounds at a high concentration were already toxic to a degree that the urea production was below 60%, the

Fig. 6. Concentration-dependent effect of troglitazone (Tro) on urea production of sandwich-cultured rat hepatocytes (SCRH) at day-4 of culture time. SCRH were incubated with different concentrations of Tro in the absence (black bars) and in the presence (gray bars) of a 60× bile acid (BA) mixture, as described in the Materials and methods section. Panel A. Concentration-dependent effect of Tro on urea production of freshly-isolated rat hepatocytes. Each bar represents mean (±SEM, n = 10) urea production in SCRH. Panel B and C. Values obtained in 2 individual batches of cryopreserved rat hepatocytes. Each bar represents mean (±SD, n = 3) urea production in SCRH. *p b 0.05, **p b 0.01 and ***p b 0.001 (Student's t-test) for pair-wise comparison between conditions in absence (black bars) and conditions in the presence (gray bars) of the 60× BA mixture.

Fig. 8. Drug-induced cholestasis index (DICI) values of different concentrations of troglitazone (Tro) in cryopreserved rat hepatocyte batches. Points represent the individual mean (±SD) DICI values obtained in 2–4 batches of sandwich-cultured rat hepatocytes (SCRH). Tro was incubated with and without bile acid (BA) mixture and DICI values were calculated based on the relative urea production. The dotted line on the Y-axis represents a DICI value of 0.8.

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M. Oorts et al. / Journal of Pharmacological and Toxicological Methods 73 (2015) 63–71

Table 4 Comparison of the Area under the Curve (AUC) values in the presence and in the absence of a bile acid (BA) mixture in freshly-isolated and cryopreserved rat hepatocyte batches. The concentration-dependent effect of Troglitazone on the urea production of sandwich-cultured rat hepatocytes (SCRH), as shown in Supplemental Fig. 3 was used to calculate the AUC values by means of the trapezoidal rule. Troglitazone

AUC (without BAs)

AUC (with BAs)

Ratio (with BAs/without BAs)

Before cryopreservation (n = 10) Batch #1; after cryopreservation Batch #2; after cryopreservation

5981 5584 6682

4019 4409 4105

0.67 0.79 0.61

resulting DICI values were excluded from further data analysis (cfr. results of 125 μM Tro). It should be noted that the DICI values determined in the present study, are the result of a single incubation period of 24 h. Hence, incubation of hepatocytes with CsA and Tro (or other compounds with a cholestatic potential) for longer periods and/or in multiple time points, could have revealed DICI values lower than 0.8 at lower concentrations of CsA and Tro. Further studies are needed to explore this possibility. CsA and Tro were chosen as test compounds, as they are known to be cholestatic in vivo, both in rat and human. CsA and Tro showed inhibition of Bsep/BSEP in vitro and resulted in a concentration-dependent decrease of DICI values obtained in SCRH and sandwich-cultured human hepatocytes (SCHH) (Akashi, Tanaka, & Takikawa, 2006; Chatterjee et al., 2014b; Funk, Ponelle, Scheuermann, & Pantze, 2001; Funk et al., 2001; Kemp, Zamek-Gliszczynski, & Brouwer, 2005; Stieger et al., 2000). Moreover, CsA is a potent inhibitor of Ntcp/NTCP and the organic anion transporting polypeptide family (Oatp/OATP), both uptake transporters of bile acids (Gertz, Houston, & Galetin, 2011; Mita et al., 2006; Schroeder et al., 1998). Therefore, the incubation of CsA in SCH could possibly lead to a protective effect from toxicity of the bile acids. Further studies can be performed to investigate this issue (i.e. pre-incubating the hepatocytes with bile acids, after which they are incubated with CsA). In case of Tro, the sulfate conjugate has been shown to be a more potent inhibitor of Bsep/BSEP, compared to the parent compound (Funk et al., 2001a). In addition, formation of reactive metabolites also plays a role in troglitazone hepatotoxicity (He et al., 2004). Overall, the results obtained in the present study did not show any significant differences in the determination of the cholestatic potential of CsA and Tro in cultures obtained from hepatocytes before and after cryopreservation, which is in concordance with literature data (Keemink, De Bruyn, Graindor, Augustijns, & Annaert, in preparation; Pang, Zaleski, & Kauffman, 1997; Santone, Melder, & Powis, 1989). Moreover, these results were confirmed by the AUC values determined from concentration-dependent profiles as depicted in the Supplemental figures. These findings can be attributed to the fact that the functionality of hepatic transport proteins and metabolizing enzymes are not compromised during/after cryopreservation. A recent study by our research group concluded no significant difference in Oatp-, Mrp2- and Ugt1mediated disposition between SCH from freshly-isolated and cryopreserved rat hepatocytes (Keemink et al., in preparation). Additionally, it has been reported that phase I and II metabolic enzymes are not compromised after cryopreservation and thawing. However, activity of hepatic uptake transporters is decreased in cultures of cryopreserved rat hepatocytes (Hewitt & Utesch, 2004; Lundquist et al., 2014). No reports were found concerning a possible alteration in the activity of hepatic efflux transporters in SRCH before and after cryopreservation. However, in case of SCHH, Bi et al., 2006 have shown that uptake and efflux transporter activities are comparable between both freshlyisolated and cryopreserved hepatocytes (Bi, Kazolias, & Duignan, 2006). In conclusion, these results suggest that freshly-isolated and cryopreserved hepatocytes can be used inter-changeably in sandwich cultures as an in vitro model to identify compounds with a potential to cause cholestasis. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.vascn.2015.03.002.

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