Supplemental material to this article can be found at: http://dmd.aspetjournals.org/content/suppl/2016/06/23/dmd.116.069831.DC1 1521-009X/44/9/1463–1479$25.00 http://dx.doi.org/10.1124/dmd.116.069831 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 44:1463–1479, September 2016 U.S. Government work not protected by U.S. copyright

Contextualizing Hepatocyte Functionality of Cryopreserved HepaRG Cell Cultures s

Jonathan P. Jackson,1 Linhou Li, Erica D. Chamberlain,2 Hongbing Wang, and Stephen S. Ferguson3

Life Technologies, Cell System Division, ADME/Tox, Durham, North Carolina (J.P.J., E.D., S.S.F.); Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland (L.L., H.W.)

Received February 1, 2016; accepted June 22, 2016

ABSTRACT Over the last decade HepaRG cells have emerged as a promising demonstrated the impact of extracellular matrix on cryo-HepaRG alternative to primary human hepatocytes (PHH) and have been functionality. Pharmacologically important drug-metabolizing al- Downloaded from featured in over 300 research publications. Most of these reports leles were genotyped in HepaRG cells and poor metabolizer alleles employed freshly differentiated HepaRG cells that require time- for CYP2D6, CYP2C9, and CYP3A5 were identified and consistent consuming culture (∼28 days) for full differentiation. Recently, a with higher frequency alleles found in individuals of Caucasian cryopreserved, predifferentiated format of HepaRG cells (termed decent. We observed liver enzyme inducibility with aryl hydrocarbon here “cryo-HepaRG”) has emerged as a new model that improves receptor, constitutive androstane receptor (CAR), and pregnane X global availability and experimental flexibility; however, it is largely receptor activators comparable to that of sandwich-cultured PHH. dmd.aspetjournals.org unknown whether HepaRG cells in this format fully retain their Finally, we show for the first time that cryo-HepaRG supports proper hepatic characteristics. Therefore, we systematically investigated CAR cytosolic sequestration and translocation to hepatocyte nuclei the hepatocyte functionality of cryo-HepaRG cultures in context with in response to phenobarbital treatment. Taken together, these data the range of interindividual variation observed with PHH in both reveal important considerations for the use of this cell model and sandwich-culture and suspension formats. These evaluations un- demonstrate that cryo-HepaRG are suitable for metabolism and covered a novel adaptation period for the cryo-HepaRG format and toxicology screening. at ASPET Journals on September 27, 2021

Introduction metabolism and toxicity, owing to their proper maintenance of metab- The liver is a major organ involved in the detoxification of both olism, transport, and receptor signaling pathways. However, the pro- endobiotic and xenobiotic chemicals. Primary human hepatocytes nounced interindividual variability and high cost of PHH has led to the (PHH) are a well accepted in vitro liver model for prediction of drug emergence of alternative cell models, such as the hepatoma-derived HepG2 and the immortalized Fa2N-4 for screening purposes. To date, these immortalized models have been associated with insufficient hepatocyte differentiation and low metabolic functionality (Hariparsad The laboratory research was funded by Life Technologies (Thermo Fisher) research and development. Resources for data analysis and manuscript et al., 2008; Donato et al., 2010). preparation were supported by the National Institutes of Health, National Institute In recent years, freshly differentiated HepaRG cells have emerged as of Environmental Health Sciences, the National Toxicology Program Division, and a promising alternative to PHH for in vitro drug-drug interaction and by Qualyst Transporter Solutions, Inc. toxicology studies. To reach phenotypic maturity, HepaRG cells grow 1Current affiliation: J.P.J. currently with Qualyst Transporter Solutions (QTS). to confluence and differentiate over 4 weeks (from progenitor cells) into 2Current affiliation: E.D.C. currently with Quest Pharmaceutical Services (QPS). cocultures of hepatocyte-like and cholangiocyte-like cells (Gripon et al., 3 Current affiliation: S.S.F. currently with the National Toxicology Program 2002). Since this model was discovered, many studies have shown that Division, National Institute of Environmental Health Sciences (NIEHS). freshly differentiated HepaRG cultures exhibit cellular interactions, drug Some of the data presented were part of a poster abstract: Jackson JP, metabolism/transport, and drug induction responsiveness comparable Edwards M, Chamberlain E, and Ferguson, SS (2011) Cryopreserved HepaRG to PHH cultures (Grime et al., 2010; McGill et al., 2011; Gerets et al., cells: an alternative in vitro screening tool for human hepatic drug metabolism, induction of metabolism, and safety applications, at the 2011 ISSX meeting; 2012; Le Vee et al., 2013; Szabo et al., 2013). A cryopreserved format Atlanta, GA. International Society for the Study of Xenobiotics, Atlanta GA. of differentiated HepaRG cells (cryo-HepaRG) has recently become dx.doi.org/10.1124/dmd.116.069831. available, improving the global availability and experimental flexibility s This article has supplemental material available at dmd.aspetjournals.org. of this model. However, the impact of detachment, cryopreservation,

ABBREVIATIONS: ABCB11, ATP-binding cassette, sub-family B, member 11; AhR, aryl hydrocarbon receptor; CAR, constitutive androstane receptor; CDCA, chenodeoxycholic acid; CDF, 5-(and-6)-carboxy-29,79-dichlorofluorescein; CDFDA, carboxy dichlorofluorescein diacetate; CITCO, 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime; cryo-HepaRG, cryopreserved HepaRG cells; DMSO, dimethyl sulfoxide; ES, estrone sulfate; EYFP-hCAR, enhanced yellow fluorescent protein–tagged hCAR; FMO, flavin monooxygenase; FXR, farnesoid X receptor; HepaRG, HepaRG cells; LC-MS/MS, liquid chromatography–tandem mass spectrometry; MRP2, multidrug resistance– associated protein 2; OATP1B1/3, organic anion–transporting polypeptides 1B1 and 1B3; PB, phenobarbital; PBS, phosphate-buffered saline; P450, cytochrome P450; PHH, primary human hepatocytes; PXR, pregnane X receptor; RIF, rifampin; SC-PHH, sandwich-cultured primary human hepatocytes; SULT, sulfotransferase; UGT, UDP-glucuronosyltransferase; WEM, Williams’ E media.

1463 1464 Jackson et al. and replating on HepaRG function has not been comprehensively needed, for each assay type .1 hour after plating, and culture media was renewed evaluated. It is known that disruption of cellular interactions during liver every 2–3 days, unless otherwise stated. All HepaRG and PHH cell culture images isolations results in PHH dedifferentiation (Godoy et al., 2013). Therefore, were captured using a Zeiss Axiovert inverted research microscope equipped with it is important to understand the consequences of detachment/reattachment phase-contrast optics, a 3CCD camera, and imaging computer with image analysis for cryo-HepaRG. To date, the effect of culture time on cryo-HepaRG software. Freshly differentiated HepaRG cells were grown and differentiated from progenitor cells as previously described (Gripon et al., 2002). metabolic competence (postreattachment to monolayers), liver enzyme PHH. Cryopreserved PHH were thawed and recovered using Cryopreserved induction, and uptake transport has not been characterized or compared Hepatocyte Recovery Media, then directly assayed in suspension form or plated with interindividual variation across large numbers of sandwich-cultured using WEM (Hepatocyte Plating Supplement Pack, serum-containing; Life primary human hepatocytes (SC-PHH) and suspensions of PHH. Finally, Technologies) at optimal density (e.g., 0.8 Â 106 cells/ml) in 24-well culture no immortalized-liver-cell-line alternative to PHH has been found to plates. Cryopreserved and fresh hepatocytes were allowed to attach for 4–6 hours properly model the constitutive androstane receptor (CAR) activation in a humidified incubator at 5% CO2 and 37°C, prior to overlay with 0.35 mg/ml pathway by which CAR is sequestered in the cytosol of hepatocytes and Geltrex in serum-free WEM supplemented with ITS+, GlutaMAX, 1% Pen/Strep, translocates to the nucleus upon activation by phenobarbital, a hallmark 15 mM HEPES buffer, and 100 nM dexamethasone. Culture media was replaced feature of functional PHH. daily. PHH reference data from lot characterizations across hundreds of donor In the current study, we evaluated cryo-HepaRG and found them to preparations (cultured and suspension formats) represent approximately equal numbers of male and female donor preparations. For Hu8033, primary hepato- resemble freshly differentiated HepaRG after 7–10 days in culture. We cytes were derived from a female donor. observed bile canaliculi formation over time, a hallmark of hepatocyte 5 CDFDA Staining. Cryo-HepaRG were thawed and plated (3 Â 10 cells/well) Downloaded from polarization operative in PHH cultures, with morphologies (i.e., cords onto 24-well culture plates using WEM supplemented with HPRG770 and of hepatocyte-like cells) stabilizing after 7–10 days in culture. We GlutaMAX. Media was replaced the next day with WEM supplemented with monitored the temporal dynamics of metabolic competence in cultured HPRG720 and GlutaMAX supplement. Media was refreshed every 2 days cryo-HepaRG and observed an adaptation period with an initial loss of thereafter. After 10 days in culture, media was aspirated from plates, and cell metabolic competence that was restored to suspension cryo-HepaRG monolayers were washed with warm Hanks’ balanced salt solution (HBSS). levels after 7–10 days in culture. Metabolic activities, liver enzyme HepaRG cultures were incubated for 15 minutes with cell culture media induction, and uptake/efflux transport in cryo-HepaRG were compared containing 5 mM CDFDA. After incubations, fluorescence photomicrographs dmd.aspetjournals.org with numerous lots of SC-PHH and suspension PHH to provide a were captured as described above with a fluorescent light source. broader context for cryo-HepaRG functionality. Our results reveal the Drug-Metabolism Assays. Cryo-HepaRG were cultured for 0 (suspension), 1, 2, 3, 4, 10, or 22 days (media was renewed every 2–3 days) in HPRG720 impact of extracellular matrix overlay on cryo-HepaRG functionality, (metabolism media supplement) and GlutaMAX prior to conducting in situ provide genotyping analysis of pharmacologically important poor incubations for metabolic activities, with the exception of induction assays (as metabolizer alleles, and demonstrate that cryo-HepaRG can properly described below). Primary human hepatocytes were cultured 48–72 hours in sequester CAR in the cytosol and translocate it to the nucleus upon WEM (ITS+, GlutaMAX, 1% Pen/Strep, and 100 nM dexamethasone) prior to phenobarbital treatment. in situ metabolism assays with probe substrates. The following final substrate at ASPET Journals on September 27, 2021 concentrations were used in cell culture media: 100 mM phenacetin (CYP1A2), 5 mM coumarin (CYP2A6), 500 mM bupropion (CYP2B6), 20 mM pacli- Materials and Methods taxel (CYP2C8), 25 mM diclofenac (CYP2C9), 250 mM S-mephenytoin Materials. Cryo-HepaRG, William’s E Media (WEM), 96-well collagen (CYP2C19), 15 mM dextromethorphan (CYP2D6), 250 mM chlorzoxazone, I-coated plates, GlutaMAX Supplement, HPRG770, HPRG720, and HPRG740 10 mM midazolam (CYP3A4/5), 200 mM testosterone (CYP3A4/5), 250 mM media supplements, Cryopreserved Hepatocyte Recovery Media, Geltrex Matrix, benzydamine (FMO), or 100 mM 7-hydroxycoumarin (sulfotransferase, UDP- Carboxy Dichlorofluorescein Diacetate (CDFDA), and PHH were obtained from glucuronosyltransferase). All in situ metabolism assays were conducted in single- Life Technologies/Thermo Fisher Scientific (Carlsbad, CA). Serum-free hepato- probe format as previously described (Jackson et al., 2009). All cell suspension cyte culture supplement ITS+ was obtained from BD Biosciences (San Jose, CA). metabolism assays were performed in single probe format as previously described Phenacetin, acetaminophen, coumarin, 7-hydroxycoumarin, bupropion, hydroxy- (Smith et al., 2012). All cryo-HepaRG metabolism assays for all substrates bupropion, paclitaxel, 6a-hydroxypaclitaxel, diclofenac, 49-hydroxydiclofenac, examined were conducted for 1 hour. PHH metabolism assay incubation periods mephenytoin, 49-hydroxymephenytoin, dextromethorphan, dextrorphan, testosterone, were substrate-dependent (15 min phenacetin, 20 min bupropion, 45 min 6b-hydroxytestosterone, midazolam, 1-hydroxymidazolam, benzydamine, benzydamine paclitaxel, 15 min diclofenac, 30 min (S)-mephenytoin, 15 min dextromethor- N-oxide, 7-hydroxycoumarin glucuronide and sulfate, omeprazole, phenobarbital phan, 15 min chlorzoxazone, 14 min testosterone, 10 min midazolam, 30 min (PB), rifampicin (RIF), dimethyl sulfoxide (DMSO), aflatoxin B1, ketoconazole, 7ethoxycoumarin, 30 min benzydamine). Metabolism assay samples were benzonapthoflavone, hyperforin, 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole- collected and stored frozen at –80°C until they were processed for liquid 5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO), taurocholate, and estrone chromatography–tandem mass spectrometry (LC-MS/MS) analysis. LC-MS/MS sulfate were obtained from Sigma-Aldrich (St. Louis, MO) or other commercial analysis of metabolism assay samples was conducted as previously described sources. (Jackson et al., 2009; Smith et al., 2012) and included standard curves with at least Culturing of Cryo-HepaRG. Cryo-HepaRG were thawed and plated (1 Â six calibration standards along with 12 quality control samples (at three different 105 cells/well) on 96-well flat-bottom collagen (I)-coated cell culture plates concentrations) dispersed at the beginning, middle, and end of analytical runs to following the Life Technologies standard plating protocol. Briefly, supplemented assess quantitative continuity throughout the run. culture media (WEM supplemented with HPRG770 and GlutaMAX) was warmed Induction Assays. To evaluate cytochrome P450 (P450) inducibility, cryo- to 37°C (water bath). Media (9 ml/plate) was transferred to a 50-ml conical tube. HepaRG were maintained in WEM supplemented with HPRG740 (no DMSO) Cryo-HepaRG were thawed at 37°C (water bath) until a small amount of ice crystal and GlutaMAX supplement beginning on day 3 of culture (initially plated in remained. Cryo-HepaRG were aseptically transferred to the 50-ml conical tube and HPRG770-supplemented media). Dosing with 50 mM omeprazole, 1 mM PB, or centrifuged for 2 minutes at 357g at room temperature. Media was aspirated and 10 mM RIF was initiated on day 3 of culture and renewed once daily over the cells were resuspended in 5 ml of HPRG770-supplemented media, and cells were ;72-hour treatment period. Inducers were dissolved in 100% DMSO and diluted counted and assessed for viability with Trypan Blue (0.05%) using a Countess 1/1000 in cell culture media for a final DMSO concentration of 0.1%. The extent (Life Technologies). Cell densities were adjusted to 1.25 Â 106 cells/ml, and 80 ml of induction was evaluated with in situ metabolism assays for CYP1A2, CYP2B6, was delivered to each well (prewetted with 45 ml of plating media) of cell-culture and CYP3A4 enzymatic activity compared with vehicle control levels. Probe plates using a multichannel pipettor. Plated cells were subsequently allowed to substrates phenacetin, bupropion, and testosterone were dissolved in 100% settle in cell culture plates for ;10 minutes in the biosafety cabinet and moved to a DMSO and applied to cell cultures (as described above) for 60 minutes. Assay humidified incubator at 5% CO2 and 37°C. Supplemented media was changed, as media was subsequently transferred to round-bottom polypropylene plates Hepatocyte Function of Cryo-HepaRG Cultures 1465

(96-well) for LC-MS/MS analysis as described earlier. For gene expression quantitatively characterized as nuclear, cytosolic, and mixed (nuclear + cytosolic) assays, established (inventoried, validated) TaqMan assays for CYP1A2, expression by counting 100 Ad/EYFP-hCAR–expressing HepaRG cells from CYP2B6, CYP3A4, and GAPDH were used. TaqMan universal PCR Master each treatment group. Mix and dNTPs were obtained from Applied Biosystems/Thermo Fisher Statistical Analyses. Multiple t-test comparisons of mean enzymatic activities Scientific (Foster City, CA) and used according to the manufacturer’s protocol. were determined in GraphPad Prism v.6.07 (GraphPad Software, La Jolla, CA) by Complementary DNA (cDNA) was generated from isolated total RNA from cell analyzing each row individually without assumption of consistent standard cultures using an ABI PRISM 6100 Nucleic Acid Prep Station and ABI chemistry deviations, correction for multiple comparisons using Holm-Sidak method, and an (ABI, Foster City, CA) following the manufacturer’s protocol for cell culture alpha of 0.05. Tukey’s pairwise comparisons of mean response data were lysates. Total RNA was quantified using a NanoDrop spectrophotometer (NanoDrop, performed after initial one-way analysis of variance using JMP 11.0.0 (SAS, NC) Wilmington, DE). A High-Capacity cDNA Archive Kit (Life Technologies) was used at an alpha of 0.05. to synthesize cDNA from 200 ng of total RNA, using random hexamers, following the manufacturer’sprotocol. Cytotoxicity Assays. For cytotoxicity assays, plating media was replaced on Results day 2 in culture with WEM supplemented with HPRG720 or HRG770 and GlutaMAX supplement. Media was refreshed every 2 days thereafter. HepaRG Culture Model Evaluation cultures were treated with aflatoxin B1 on day 7 in culture. Twenty-four hours Hepatocyte morphology is known to be a useful indicator of mono- after treatment, ATP content assays were performed. ATP levels were determined layer integrity/differentiation status with in vitro liver models (Hamilton using CellTiter-GLO Luminescent Cell Viability Assay according to the manufac- et al., 2001). Therefore, we assessed cryo-HepaRG culture morphologies ’ turer s instructions (Promega Corporation, Madison, WI) using a FLUOstar Omega compared with freshly differentiated HepaRG and SC-PHH (Fig. 1, A Downloaded from luminometer (BMG Labtech, Cary, NC). Dose-response curves were modeled with and C). In initial cell biology evaluations with cryo-HepaRG, we observed the Hill equation, as previously described (Beam and Motsinger-Reif, 2011). a rapid attachment (,10 minutes) to cell culture plates compared with the Uptake Transport Assays. Plated uptake assays were performed on day-4 of ; – – cryo-HepaRG culture. Cell-culture media was aspirated from culture plates, and 4 6 hours generally required for PHH. After 1 2 hours in culture, cryo- cell monolayers were washed three times with warm HBSS plus HEPES buffer. HepaRG appeared rounded (Fig. 1D) and lacked characteristic cobblestone Cells were preincubated with the third wash in a 37°C humidified incubator with appearance and cord-like networks of hepatocyte-like cells observed with

95% air/5% CO2 for 10 minutes. Buffer was subsequently replaced with dosing fully/freshly differentiated HepaRG (Fig. 1A). Cobblestone-like networks dmd.aspetjournals.org solution containing varying concentrations of 3H-radiolabeled taurocholate, are common to epithelial cells in confluent monolayers that autoassemble, estradiol-17b-glucuronide, or estrone sulfate prepared in HBSS plus HEPES resembling structures found in vivo within the liver (Godoy et al., 2013). buffer, and incubated for 30 minutes. After incubation, dosing solutions were Their absence after only a few hours in culture indicates that cryo-HepaRG aspirated and HepaRG or PHH cultures were washed three times with ice-cold were undergoing a significant transition to their new culture environment. – ° buffer and placed at 80 C for a minimum of 15 minutes prior to cell lysis with While cryo-HepaRG began to resemble fully differentiated HepaRG ;24 0.1% Triton X solution. Samples were analyzed using a MicroBeta TriLux hours postattachment (Fig. 1E), 7–10 days were required for full recovery scintillation counter (Perkin Elmer, Waltham, MA). Primary hepatocyte uptake of morphology (Fig. 1, F and B). Initial experiments to optimize cryo- transport assays were performed analogously to HepaRG cultures, with the at ASPET Journals on September 27, 2021 exception that 10-minute incubations were performed, and cultures were HepaRG cell seeding densities using metabolic competence (Supplemental maintained for 18–24 hours (postplating), prior to uptake assays. Fig. 1) and cell morphologies resulted in selection of ;100,000 cells/well CYP3A4 Immunohistochemistry. CYP3A4 antibody was purchased from (96-well format). Seeding densities greater than ;100,000 cells/well Abcam (Cambridge, MA). Treated cell cultures (24-well) were washed with resulted in overcrowding of monolayers and forced HepaRG cells to phosphate-buffered saline (PBS), fixed with 500 ml of 2% paraformaldehyde intermittently rise out of plane. Analogously to PHH cultures (LeCluyse solution with 0.1% saponin in PBS for 30 minutes at room temperature. Fixed et al., 1994) and freshly differentiated HepaRG (Antherieu et al., 2010), cells were washed three times with PBS for 1 minute on a micro-orbital shaker cryo-HepaRG appeared to form bile canalicular structures after ;3daysin ; m ( 200 rpm). Subsequently, blocking was performed with 300 l of Image-iT FX culture). In general, these structures were more rounded between multiple Signal Enhancer (Thermo Fisher Scientific) and incubated at room temperature cells compared with longer linear or y-like structures common to SC-PHH. overnight. Blocked cells were washed three times with 500 ml/well of warm PBS Bile canalicular formation was further evaluated using CDFDA staining. for 1 minute on an orbital shaker (;200 rpm). Cells were further incubated with primary antibody at 1/200 dilution for 1 hour at 37°C and imaged with a Zeiss CDFDA is converted to CDF in hepatocytes, and CDF is a substrate of bile – Axiovert inverted research microscope as described earlier. efflux transporter multidrug resistance associated protein 2 (MRP2) in Protein Quantification Assays. Protein quantification assays were performed liver (Hoffmaster et al., 2004). MRP2 and other apical efflux transporters using Thermo Scientific Pierce BCA Protein Assay Kit following the manufac- are generally thought to be internalized upon disruption of cell-cell turer’s instructions, and absorbance was quantified using a FLUOstar Omega interactions during isolation of PHH. Under suitable culture conditions, plate reader (BMG Labtech). hepatocytes repolarize in culture and canalicular transporters (i.e., MRP2) Genotyping Analysis. Genomic DNA was isolated from HepaRG cells migrate back to the canalicular membrane over time (2–4days).CDFDA utilizing an ABI PRISM 6100 Nucleic Acid Prep Station and ABI chemistry staining of cryo-HepaRG cultures demonstrated clear localization of CDF ’ following manufacturer s instructions (Life Technologies). Genotyping of between hepatocytes that perfectly overlie with canalicular domains in HepaRG genomic DNA was conducted using TaqMan Drug Metabolism phase contrast images (Figs. 1, G–I). Approximately 3 days in culture were Genotyping Assays on an ABI 7900 Real-Time PCR instrument following required to form bile canaliculi in cryo-HepaRG. Collectively these manufacturer’s instructions (Thermo Fisher Scientific). Translocation of CAR in HepaRG Cells. An adenovirus-expressing observations demonstrate that cryo-HepaRG repolarize over time in enhanced yellow fluorescent protein–tagged hCAR (Ad/EYFP-hCAR) construct culture analogously to PHH isolated from human livers and do not was generated and functionally characterized, as previously reported (Li et al., immediately reform fully differentiated monolayers. 2009a). Cryo-HepaRG were thawed and seeded on 24-well BioCoat plates (Thermo Fisher) in WEM containing HepaRG Induction Media Supplements Phase I and Phase II Drug-Metabolism Competence Characterization HPRG740 (serum-containing) and HPRG750 (serum-free). Twenty-four hours and Temporal Dynamics after plating, HepaRG cells were infected with Ad/EYFP-hCAR (5 ml virus per well) for 12 hours, followed by treatment with vehicle control (0.1% DMSO) or As a marker of hepatocyte differentiation, we evaluated the metabolic PB (1 mM) for an additional 8 hours. Treated cells were then subjected to confocal competence of cryo-HepaRG after 10 days in culture with a panel of microscopy analysis with a Nikon C1-LU3 instrument attached to an inverted 12 pharmacologically important drug-metabolizing enzymes (Table 1). Nikon Eclipse TE2000 microscope (Tokyo, Japan). Localization of hCAR was Enzymatic activities [CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, 1466 Jackson et al. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 27, 2021

Fig. 1. Morphologic evaluation, seeding density optimization, and bile canaliculi staining. Cell morphology photomicrographs with HepaRG cell cultures at various stages of culture. (A) Freshly differentiated HepaRG after ;4 weeks in culture. (B) Cryo-HepaRG after 10 days in culture (postplating). (C) Sandwich cultures of cryopreserved PHH (3 days in culture). (D) Cryo-HepaRG in culture ;6 hours postplating. (E) Cryo-HepaRG ;24 hours in culture. (F) Cryo-HepaRG ;7 days in culture. (G) Phase contrast bright-field image of cryo-HepaRG matched to CFDFA staining (H), and the merged image (I) highlighting MRP2 efflux function indicative of a polarized monolayer functional for biliary efflux.

CYP2C19, CYP2D6, CYP2E1, and CYP3A4/5, flavin monooxygenase observed with 52 donor preparations of SC-PHH (1.47–178 pmol/min (FMO), UDP-glucuronyltransferase (UGT), and sulfotransferase million cells, mean of 55.2 6 49.4). Although initially appearing super- (SULT)] were determined in situ and compared with the ranges of physiologic, this level of CYP3A4/5 activity was lower than median interindividual variation observed in SC-PHH under comparable assay testosterone 6b-hydroxylase activity observed across 195 suspensions conditions. Following 10 days of culture, CYP1A2 activity (phenacetin of PHH (407 pmol/min million cells). Consistent with the elevated O-deethylase) was observed at 2.78 6 0.728 pmol/min-million cells CYP3A4/5 basal activity observed across 52 cryo-HepaRG prepara- in cryo-HepaRG compared with a mean activity of 7.18 6 7.78 (near tions, CYP2B6 activity levels also exceeded the range observed with 25th percentile, Fig. 2A in red) compiled from a panel of 52 donor SC-PHH (P , 0.05) at 17.9 pmol/min-million cells compared with preparations of SC-PHH. Cryo-HepaRG CYP1A2 mean activity was 0.21–13.1 pmol/min-million cells. Although CYP3A4 and CYP2B6 statistically different from the SC-PHH mean activity, but the magnitude exhibited disproportionately high levels of activity in cryo-HepaRG, it is was less than 2-fold from the median CYP1A2 activity of the 52 different important to note that these enzymes are also among the most inducible SC-PHH preparations. Notably, both plated cryo-HepaRG and SC-PHH drug-metabolizing enzymes in human liver. Less inducible enzymes fall profoundly short of in vivo-like levels at ;6% of median CYP1A2 such as CYP2A6, CYP2C8, CYP2C9, CYP2C19, and CYP2D6 were activity observed across 212 PHH suspension preparations evaluated not statistically different (P , 0.05) from the mean activities of 52 SC- (48.0 pmol/min-million cells), which of course, assumes PHH suspensions PHH preparations evaluated (Table 1). Generally, all activities measured approximate in vivo levels. in situ were around 10% of the median activities observed with PHH CYP3A4/5 activity in cryo-HepaRG was evaluated utilizing two suspensions. CYP2D6 activity in cryo-HepaRG was near the low end of probe substrates (testosterone and midazolam) following 10 days in the range compared with SC-PHH and is further discussed below in culture under metabolism media (day-10). Testosterone 6b-hydroxylase Genotyping of Important Human Absorption, Distribution, Metabolism, activity was 248 6 50.9 pmol/min-million cells in cryo-HepaRG, and Excretion Genes HepaRG. CYP2E1 activity was also evaluated in whereas midazolam 1-hydroxylase activity was 28.4 6 2.39 pmol/min- cryo-HepaRG cultures under these conditions (data not shown); million cells. Interestingly, the testosterone 6b-hydroxylase activity in however, the potent inhibitory effects of DMSO on CYP2E1 prohibited cryo-HepaRG was ;6.8-fold higher than median and was significantly accurate evaluation of CYP2E1 metabolic competence. Enzymatic higher (P , 0.0001) than the mean, median, and top of the range activity data for FMO, UGT, and SULT were also determined for Hepatocyte Function of Cryo-HepaRG Cultures 1467

TABLE 1 Baseline drug-metabolizing enzyme activity at day-10 (metabolism media) with cryo-HepaRG versus sandwich-culture and suspension PHH Cryo-HepaRG cells were cultured for 10 days postplating in HepaRG metabolism media–supplemented culture media in eight replicate wells. Primary human hepatocytes were directly assayed or cultured for 4–5 days in serum-free induction media with ITS+, 100 nM dexamethasone, 15 mM HEPES, 1% pen/strep. Drug-metabolism enzyme–specific activities were evaluated using in situ incubations with probe substrates in cryopreserved human hepatocyte culture. LC-MS/MS methods were used to quantify specific metabolite concentrations. Means were compared using unpaired t tests (i.e., Welch’s correction).

Mean Difference Mean Statistical PHH Suspension Mean Specific (.2-fold) HepaRG Enzyme Substrate Marker Metabolite Cell Culture Model Difference HepaRG Median Specific Activity 6 S.D. versus versus SC-PHH Activities SC-PHH Median

pmol/min-million cells pmol/min-million cells CYP1A2 Phenacetin Acetaminophen HepaRG 2.78 6 0.728 P , 0.0001 No 48.0 (212 donor preps) SC-PHH (N=52) 7.18 6 7.78 CYP2A6 Coumarin 7-Hydroxycoumarin HepaRG 0.851 6 0.159 No No Not Available SC-PHH (N=53) 1.03 6 0.974 CYP2B6 Bupropion Hydroxybupropion HepaRG 17.9 6 2.21 P , 0.0001 Yes, 8.3X 32.1 (137 donor preps) SC-PHH (N=52) 2.94 6 2.78 CYP2C8 Paclitaxel 6a-Hydroxypaclitaxel HepaRG 0.223 6 0.035 No No 5.24 (149 donor preps) SC-PHH (N=34) 0.517 6 0.802 CYP2C9 Diclofenac 49Hydroxydiclofenac HepaRG 3.94 6 0.380 No No 88.2 (187 donor preps) 6

SC-PHH (N=54) 5.37 9.54 Downloaded from CYP2C19 Mephenytoin 49-Hydroxydiclofenac HepaRG 1.52 6 0.408 No No 13.8 (208 donor preps) SC-PHH (N=48) 2.26 6 4.71 CYP2D6 Dextromethorphan Dextrorphan HepaRG 0.401 6 0.0622 No, P = 0.057 No 21.1 (24 donor preps) SC-PHH (N=36) 1.18 6 1.70 CYP3A4/5 Testosterone 6b-Hydroxytestosterone HepaRG 248 6 50.9 P , 0.0001 Yes, 6.8X 407 (195 donor preps) SC-PHH (N=52) 55.2 6 49.4 CYP3A4/5 Midazolam 1-Hydroxymidazolam HepaRG 28.4 6 2.39 NA NA 63.7 (132 donor preps) SC-PHH (N=0) Not available dmd.aspetjournals.org FMO Benzydamine Benzydamine N-oxide HepaRG 17.3 6 1.74 NA NA 144 (105 donor preps) SC-PHH (N=0) Not available UGT 7-Hydroxycoumarin 7-Hydroxycoumarin HepaRG 346 6 49.7 NA NA 494 (196 donor preps) glucuronide SC-PHH (N=0) Not available SULT 7-Hydroxycoumarin 7-Hydroxycoumarin HepaRG 9.23 6 2.92 NA NA 37 (195 donor preps) sulfate SC-PHH (N=0) Not available

NA, Data from SC-PHH not available. at ASPET Journals on September 27, 2021 cryo-HepaRG and reported in Table 1. While no FMO, UGT, or SULT To further characterize the temporal dynamics of metabolic compe- data were available in SC-PHH under these assay conditions, cryo- tence with cryo-HepaRG cells, we generated specific activity data with HepaRG exhibited ;10% FMO and ;30% SULT activity levels suspensions of cryo-HepaRG in their initial form for comparison with compared with PHH suspensions, and UGT activity levels were cultured cryo-HepaRG at day-4 and day-10 (Table 2). In general, comparable between cryo-HepaRG and PHH suspensions. cryo-HepaRG suspensions and day-10 cultures of cryo-HepaRG (in As demonstrated in Table 1, SC-PHH lose a substantial proportion of HPRG720 metabolism media) produced comparable metabolic activi- drug-metabolism competence over time in culture from their initial ties across nine metabolizing enzymes, consistent with cell morphology suspension format. We hypothesized that with cryo-HepaRG a similar observations. A good correlation (Pearson correlation with R = 0.998) of phenomenon may occur that has not been reported previously and may enzymatic activities was observed between cryo-HepaRG suspensions impact metabolism-related research findings. Therefore, we compared and day-10 cultures of cryo-HepaRG (Fig. 3A). These data coupled with CYP1A2, CYP2B6, and CYP3A4/5 enzymatic activities of day-4 cryo- the day-4 enzymatic activity data suggest that cryo-HepaRG cells (under HepaRG (Table 1) with day-10 cryo-HepaRG against the activities metabolism media) initially lose much of their metabolic competence observed across 52 SC-PHH preparations (Fig. 2, A–C). Here we but recover to suspension-like levels over ;10 days. For the cyto- observed that day-4 CYP1A2 activity in cryo-HepaRG fell below the chromes P450 evaluated, day-10 metabolic competence was on average 25th percentile of the SC-PHH range compared with day-10 activity that 8.6-fold higher than day-4, while FMO, UGT, and SULT activities were rose ;3-fold to levels near the 25th percentile of SC-PHH (Table 2, 2.0-, 3.8-, and 1.8-fold higher on day-10 relative to day-4, respectively. We Fig. 2A). CYP2B6 and CYP3A4 showed similar trends, shifting from extended this study out to 22 days with activity data shown in Fig. 3C that lower activities at day-4 (near the 25th percentile and average of the were normalized to respective day-4 activities in Fig. 3D. Metabolic distributions, respectively) to levels above the SC-PHH range by day-10, competence appeared to achieve maximal levels at ;10 days in culture with increases of 19.7-fold and 3.8-fold, respectively (Fig. 2, B and C). across the metabolic pathways assessed and was largely maintained at One possible explanation for the observed increases in P450 activity comparable levels until day-22. Notably, day 16 enzymatic activities (normalized to cell numbers plated) over time in culture could be cellular were lower than metabolic activities on day-10 and day-22 across the panel proliferation occurring between day-4 and day-10. Although prolifera- of enzymes. It is not clear whether this decrease was biologically important tion is improbable in contact-inhibited confluent monolayers (Gripon as monolayer morphology appeared unchanged after day-10 and time et al., 2002), we measured total protein content over time as a marker of points on both sides of day 16 were generally comparable. cell number and observed no statistically significant changes in total As a result of the observed lower P450 activities with day-4 cryo- protein content across the time points examined (Supplemental Fig. 1). HepaRG, we further evaluated the loss of activity from suspensions Therefore, increased drug-metabolism competence over time along with through the first 96 hours in culture. As hypothesized, cryo-HepaRG morphologic observations appears to reflect an improved hepatocyte cultures appear to undergo a loss of activity (;10-fold for CYP3A4) differentiation status rather than an increase in the number of HepaRG similar to that observed with PHH (Smith et al., 2012) as they transition cells. from suspension to cell culture monolayers after removal from the liver 1468 Jackson et al.

To more comprehensively contextualize cryo-HepaRG metabolic competence, we calculated cryo-HepaRG day-4, cryo-HepaRG day-10, and SC-PHH day-4 enzymatic activities as percentages of mean suspension PHH activities (Fig. 4). From these data, PHH suspension activities far exceeded (P , 0.05) those observed with SC-PHH for all enzymes investigated. This was also true for day-4 cryo-HepaRG. For day-10 cryo-HepaRG, mean activities were also lower and generally statistically significant (P , 0.05) compared with mean and median PHH suspension activities. However, owing to high interindividual variability observed in CYP3A4/5 (P = 0.058 for 1-hydroxymidazolam and P = 0.064 with 6bT), CYP2C19 (P = 0.19), and UGT (P = 0.15) activities, statistical significance was not reached at the P , 0.05 level. These data also show significant (P , 0.05) increases for all evaluated enzymatic activities from day-4 to day-10 with cryo-HepaRG cultures in metabolism media. Day-10 cryo-HepaRG were also statistically (P , 0.05) indistinguishable from SC-PHH across this panel of metabolic activities with the exception of the higher observed activities Downloaded from of CYP2B6 and CYP3A4/5 as described earlier. Taken together, these data demonstrate that day-10 cryo-HepaRG cultures were largely compa- rable to SC-PHH with elevated levels of CYP2B6 and CYP3A4/5, whereas day-4 cryo-HepaRG were generally lower in metabolic capacity than SC-PHH with the exception of CYP3A4/5 (6bT). dmd.aspetjournals.org Effects of Overlay with Extracellular Matrix on Metabolism and Cell Culture Morphologies Overlay of PHH cultures with extracellular matrices (e.g., Geltrex, Matrigel) in “sandwich” configuration is standard practice with PHH to improve longevity, cuboidal three-dimensionality, and hepatocyte differentiation (LeCluyse et al., 1994). Therefore, we assessed the impact of Geltrex overlay on cultures of cryo-HepaRG cells in serum- at ASPET Journals on September 27, 2021 free HPRG750-supplemented induction media (contains no DMSO). Figure 5, A and B, show cryo-HepaRG cell morphology photomicro- graphs with and without Geltrex overlay (0.35 mg/ml). Overlay of cryo- HepaRG cultures led to a marked change in cell morphologies, producing improved homogenous monolayers within a single focus plane. Overlay appeared to shift the cell populations toward more three- dimensional morphologies analogous to SC-PHH (i.e., smaller nuclear- Fig. 2. HepaRG metabolic competence contextualized to PHH sandwich cultures. to-cytosolic ratios), and qualitatively appeared to produce higher Box and whisker plots show ranges of metabolic competence for CYP1A2 (A), proportions of hepatocyte-like cells (fewer cholangiocyte-like cells). CYP2B6 (B), and CYP3A4/5 (C) from PHH sandwich cultures (52 donor These characteristics are generally associated with improved hepatocyte preparations) with violet diamonds representing cryo-HepaRG metabolic activity at day-4 and red diamonds representing day-10 cryo-HepaRG. Error bars show functionalities; therefore, we further evaluated the effects of overlay on standard deviations of eight replicate wells from a single lot, and box and whiskers metabolic competence. CYP1A2, CYP2B6, and CYP3A4 were exam- show the PHH distribution (52 donor preparations) including: minimum/maximum ined in situ, and the results are shown in Fig. 5C. Approximately 2- to of the range, mean (crosshair), 25th and 75th percentiles, and median of the distribution. Data were generated using probe substrates phenacetin, bupropion, and 3-fold increases in basal metabolic activities were observed in the testosterone for CYP1A2, CYP2B6, and CYP3A4/5, respectively. LC-MS/MS presence of Geltrex overlay consistent with cell morphology observa- methods were used to quantify metabolite concentrations from in situ incubations of tions. To further evaluate the effects of Geltrex overlay on cryo-HepaRG spent culture media to calculate specific activities. Statistical significance (multiple t cultures, immunohistochemistry was performed for CYP3A4, a marker tests, P , 0.05) between SC-PHH and cryo-HepaRG indicated with an asterisk (*). of differentiated hepatocytes (Fig. 5, D–G). Immunohistochemistry for CYP3A4 was able to effectively stain hepatocyte-like HepaRG cells in standard cryo-HepaRG cultures (Fig. 5, D versus E). Upon overlay with (Fig. 3B). The observed “half-life” of CYP3A4/5 testosterone 6b- Geltrex, the CYP3A4 staining became more prevalent across the culture hydroxylase activity was 34.1 hours 6 1.02 hours, which is consistent with increased fluorescence coverage overall and larger areas of with the time frame observed with PHH (;29 hours) (Smith et al., CYP3A4 staining (Fig. 5, F and G). Taken together, these data indicate 2012). Minimum activity level was observed after ;72 hours in culture, that overlay of cryo-HepaRG improved their hepatocyte functionality as later time points appeared to produce higher activities. In summary, under the conditions examined. physiologically relevant levels of drug-metabolism activities were observed in cryo-HepaRG cultures compared to SC-PHH. However, cryo-HepaRG metabolic competence did not appear to be a static, Genotyping of Important Human Absorption, Distribution, intrinsic property of cryo-HepaRG postdifferentiation/cryopreservation, Metabolism, and Excretion Genes HepaRG as cryo-HepaRG metabolism levels exhibit marked temporal dynamics CYP2D6 and CYP2C9 enzymatic activities (Table 1) in cryo- over the initial 10 days in culture, which is important to understand in HepaRG cultures yielded lower levels of metabolic activity (near the metabolism-related research. lower end of the ranges of interindividual variation) compared with Hepatocyte Function of Cryo-HepaRG Cultures 1469

TABLE 2 HepaRG metabolic competence summary: suspension HepaRG versus cultures (metabolism media)

Enzyme (Marker Metabolite) HepaRG Suspension Activity 6 S.D. HepaRG Day-4 Cultures Activity 6 S.D. HepaRG Day-10 Cultures Activity 6 S.D.

pmol/min-million cells CYP1A2 (APAP) 1.26 6 0.0896 0.736 6 0.135 2.78 6 0.728 CYP2A6 (7OHCMN) 2.92 6 0.128 0.0905 6 0.0872 0.850 6 0.160 CYP2B6 (OHBP) 6.95 6 0.306 0.908 6 0.160 17.9 6 2.21 CYP2C8 (6a-OHTAX) 0.235 6 0.0173 0.0812 6 0.0539 0.223 6 0.035 CYP2C9 (4OHDC) 4.89 6 0.382 0.437 6 0.0785 3.94 6 0.380 CYP2C19 (4HMPN) 1.27 6 0.0404 Not determined 1.52 6 0.408 CYP3A4/5 (OHMDZ) 25.6 6 0.586 4.01 6 0.588 28.4 6 2.39 CYP3A4/5 (6bT) 377 6 13.7 65.1 6 8.44 248 6 50.9 FMO (BNO) 10.4 6 0.499 8.48 6 1.77 17.3 6 1.74 UGT (7OHCMN-Glc) ND 90.9 6 8.22 346 6 49.6 SULT (7OHCMN-Sult) ND 5.08 6 1.02 9.23 6 2.91

ND, Not determined.

SC-PHH. HepaRG cells are known to contain only a single copy of presence of multiple P450 single-nucleotide polymorphisms (summa- Downloaded from chromosome 22, which encodes for CYP2D6 and could contribute to rized in Table 3). As suspected from enzymatic activity data, HepaRG the lower CYP2D6 activity (Gripon et al., 2002). However, low levels cells contain poor metabolizer alleles for CYP2D6 (*2 and *9). These of CYP2D6 and CYP2C9 could arise from the presence of poor results suggest that HepaRG cells are deficient in CYP2D6 metabolism. metabolizer alleles in cells originating from a female Caucasian donor. The small observable dextromethorphan conversion to dextrorphan Therefore, we assessed common poor metabolizer alleles in a panel of could be the product of other enzymes including CYP3A4/5 as previ- drug-metabolizing enzymes (CYP2C9, CYP2C19, CYP2D6, CYP3A5) ously reported (Yu and Haining, 2001). Our genotyping analysis also dmd.aspetjournals.org known to be important for interindividual variation in drug clearance revealed poor metabolizer alleles for CYP2C9 (*2/*2) consistent with (Ingelman-Sundberg et al., 2007). Genotyping analyses revealed the the observed low CYP2C9 activity. For CYP3A5, HepaRG cells at ASPET Journals on September 27, 2021

Fig. 3. Metabolic competence (Phase I and Phase II) comparisons over time in culture. Cryo-HepaRG cultures were evaluated for metabolic competence at various times in culture over a range of specific activities with a panel of probe substrates reflective of various metabolic pathways (P450, FMO, UGT, and SULT). Cryo-HepaRG were thawed and plated using HPRG770-supplemented media then switched and maintained in HPRG720-supplemented media (metabolism media) after ;24 hours in culture in sandwich cultures on collagen (Type I) coated plates. (A) Pearson correlation analysis relating observed specific activities from suspension cryo-HepaRG (preplating) compared with day-10 cultures of cryo-HepaRG over a range of Phase I and Phase II drug-metabolizing enzymes. (B, C) Evaluation of the temporal dynamics of cryo- HepaRG activity over time in culture. (D) Loss of CYP3A4/5 with cryo-HepaRG in culture during the initial days of culture, fit in GraphPad Prism 6.07 using nonlinear regression. Data represent mean and standard deviation of eight independent replicate wells (A–C) and nine independent replicate wells in (D). 1470 Jackson et al. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 27, 2021

Fig. 4. Panel of liver enzyme activities characterizing cryo-HepaRG versus PHH: Metabolic activities with cryo-HepaRG cultures and SC-PHH were contextualized to mean activities from ranges of interindividual variation observed with suspensions of PHH. Data represent mean and standard deviation percentages of PHH suspension activities (mean) reflective of liver-like levels of enzymatic activities. Statistical significance (multiple t tests, P , 0.05) between mean suspension PHH enzymatic activities versus other models indicated via asterisk (*). contained two CYP3A5*3 alleles. These are known to be null alleles P450 basal activities, produced a shift in aflatoxin B1 potency consistent owing to expressed RNA instability and are present in the majority of a metabolically-activated toxicity (Fig. 6C). These data highlight the individuals of Caucasian descent. Therefore, it is probable that CYP3A4/5 utility of the cryo-HepaRG culture model for assessing metabolically activity in HepaRG cells is predominantly attributable to CYP3A4 activated toxicity, supporting previous studies that showed the utility of activity. P450 inhibitors for studying mechanisms of toxicity (Li, 2009b), and replicating previous studies that demonstrated freshly differentiated Metabolism-Dependent Cytotoxicity in Cryo-HepaRG Cultures HepaRG are a sensitive model in which to study aflatoxin B1 cytotoxicity Aflatoxin B1, a well known metabolism-dependent hepatotoxicant, (Aninat et al., 2006). has been shown to undergo metabolic activation via CYP3A4 and CYP1A2 producing a highly reactive epoxide metabolite (Essigmann Active Uptake Transport Characterization et al., 1977). The well documented mechanism of action of aflatoxin B1 An important and often rate-limiting step in drug clearance is hepatic makes it an attractive probe toxicant for evaluating the utility of cryo- uptake transport (Shitara et al., 2013). Active uptake transport function HepaRG to support evaluation of metabolism-dependent toxicity. was evaluated in cultures of cryo-HepaRG using tritium-labeled HepaRG cells were cultured in HPRG720 metabolism media for 7 days taurocholate [a sodium/taurocholate cotransporting polypeptide (NTCP) prior to dosing cultures with aflatoxin B1, ketoconazole (a reversible substrate], estrone sulfate [an organic anion–transporting polypeptides inhibitor of CYP3A metabolism in humans), or the combination of these 1B1 and 1B3 (OATP1B1/3) substrate], and estradiol-17b-glucuronide two chemicals. Cryo-HepaRG cultures were treated for ;24 hours prior (OATP1B1/3). For these evaluations HepaRG cells were cultured for to cell health/stress evaluations (Fig. 6). Both lactate dehydrogenase 10 days prior to initiating experiments. Active uptake with taurocholate and ATP data confirm aflatoxin B1-related cytotoxicity at 5–10 mM was assessed at multiple concentrations at both 4°Cand37°C to estimate half-maximal effective concentrations (EC50). Ketoconazole (20 mM) the respective passive and active uptake transport capacity of cultured was able to attenuate the observed cytotoxicity (Fig. 6, A and B). cryo-HepaRG (Fig. 7A) with representative uptake transport data from a Comparison of aflatoxin B1 cytotoxicity under HPRG720 versus single SC-PHH preparation (Hu8083) shown in Fig. 7B. For cryo- HPRG730 (“Tox” media) media supplements, known to modulate HepaRG, active uptake transport at 37°C was readily distinguishable Hepatocyte Function of Cryo-HepaRG Cultures 1471 Downloaded from dmd.aspetjournals.org at ASPET Journals on September 27, 2021

Fig. 5. Cell morphologies and metabolic competence of cryo-HepaRG with Geltrex overlay. Cryo-HepaRG were cultured without (A) or with (B) Geltrex overlay (0.35 mg/ml) yielding a more coplanar culture in the presence of Geltrex overlay and more cuboidal cell morphologies consistent with a more three-dimensional configuration. (C) After 4 days in culture, metabolic competence for CYP1A2, CYP2B6, and CYP3A4/5 was assessed in cryo-HepaRG with and without Geltrex overlay in HPRG750-supplemented serum-free culture media. (D) Phase contrast image of HepaRG without Geltrex overlay matched to (E) immunohistochemistry for CYP3A4-immunoreactive protein without Geltrex overlay after 7 days in culture in HPRG720-supplemented “metabolism” media. (F) Phase contrast of image of HepaRG with Geltrex overlay matched to (G) immunohistochemistry for CYP3A4-immunoreactive protein with Geltrex overlay after 7 days in culture in HPRG720-supplemented “metabolism” media. Data represent mean of three independent replicate wells, and error bars represent the standard deviations of the respective mean responses. Asterisk (*) indicates P , 0.05 (Tukey’sPairwise Comparison) relative to respective No Overlay control. from uptake evaluated at 4°C, consistent with competence for active SC-PHH data were not available for ES uptake. However, median uptake via NTCP. The estimated Km for taurocholate uptake was E2-17bG(1mM) uptake (37°C) across 76 lots of SC-PHH preparations 21.1 mM 6 3.08 mM for cryo-HepaRG compared with 6.68 6 0.564 was 2.92 pmol/min per milligram compared with 0.357 6 0.0181 pmol/min for Hu8083 SC-PHH fit to a Michaelis-Menten model. The extent of per milligram (0.5mM) under comparable culture conditions with cryo- taurocholate uptake in cryo-HepaRG cultures appeared to be somewhat HepaRG (Table 4). The presence of serum in cell culture media did lower than in SC-PHH, but it is notable that the percentages of appear to elevate uptake transport with both ES and E2-17bG. Geltrex hepatocytes in these models are different. To further contextualize the overlay of cryo-HepaRG cultures did not appear to have a profound extent of taurocholate uptake transport in cryo-HepaRG cultures, we effect on uptake transport, but uptake did appear marginally higher in the plotted 1 mM taurocholate uptake transport rates versus the range of presence of overlay. Collectively these data demonstrate that cryo- transport rates across 76 donor preparations of SC-PHH (37 female, HepaRG cultures were competent for active uptake transport but have 39 male) under the analogous assay conditions (Fig. 7C). Cryo-HepaRG a somewhat reduced capacity compared with SC-PHH under the culture uptake was comparable to SC-PHH, falling above the 25th percentile of conditions examined. the distribution (9.3 pmol/min per milligram) but lower than the median Functional Xenobiotic Signaling Pathways in Cryo-HepaRG. rate of SC-PHH taurocholate update (11.8 pmol/min per milligram). We Hepatic cell lines such as HepG2 and Fa2N-4 have been used in further evaluated cryo-HepaRG uptake transport competence with screening and mechanistic research to represent hepatic function in vitro. estrone sulfate (ES) and estradiol-17b-glucuronide (E2-17bG), known However, numerous reports have demonstrated the severely limited substrates for OATP uptake transporters OATP1B1 and OATP1B3 differentiation status for mature hepatocyte phenotypes in these cell (Fig. 7, D and E, Table 4). Cryo-HepaRG cultures produced some lines, including xenobiotic metabolism and receptor signaling pathways active uptake with estrone sulfate at both 0.5 and 40 mM concentrations (Donato et al., 2008; Hariparsad et al., 2008). Therefore, we evaluated (Fig. 7, D and E). Lower proportions of active uptake were observed cryo-HepaRG cultures with various selective activators of aryl hydro- with E2-17bG(;2-fold over 4°C control, data not shown). Reference carbon receptor (AhR), CAR, pregnane X receptor (PXR), and farnesoid 1472 Jackson et al.

TABLE 3 in situ metabolism assays. Concentration-related increases in CYP1A2 Genotyping summary for ADME genes in HepaRG enzymatic activity were observed with the AhR agonist omeprazole. These responses were compared to ranges of induction responses Gene Polymorphism Assessed Genotype in HepaRG observed in cultures of PHH and generally showed comparable (and P450 CYP2C9*2 *2/*2 substantially less variable) induction well within the ranges of “normal” CYP2C9*3 WT/WT response (Fig. 9). Additionally, CYP1A2 mRNA was induced over three CYP2C9*6 WT/WT CYP2C19*2 WT/WT independent assay plates with an average of ;350-fold over 0.2% CYP2C19*3 WT/WT DMSO control (Fig. 10A). Induction of CYP1A2 mRNA was larger CYP2C19*6 WT/WT (fold over control) compared with enzymatic activity responses, a CYP2D6*2 *2/WT CYP2D6*3 WT/WT phenomenon common to SC-PHH (unpublished observations). It is also CYP2D6*4 WT/WT notable that for induction assays, metabolism incubations with phenac- CYP2D6*6 WT/WT etin required ;1 hour to produce sufficient levels of the CYP1A2- CYP2D6*9 *9/WT CYP3A5*3 *3/*3 specific metabolite acetaminophen. This is comparable to the incubation CYP3A5*6 WT/WT time frame (30–60 minutes) effective with SC-PHH that produces robust CYP3A5*8 WT/WT activity levels yet avoids substrate depletion. By comparison, ;24 hours a Transporter OATP1B1*5 *5/WT are commonly required to observe specific metabolite levels with OATP1B3 (T334G) WT/WTa dedifferentiated cell lines such as HepG2 (Granata et al., 2006). Downloaded from ADME, absorption, distribution, metabolism, and excretion; WT, the wildtype allele was Induction of CYP2B6 is known to be a marker of CAR and PXR observed in HepaRG cells. aData reported by BioPredic International (Saint-Grégoire, France) on Certificates of Analysis. activation, with both receptors activating response elements within the CYP2B6 upstream regulatory regions (Wang et al., 2003). Although X receptor (FXR), monitoring induction of downstream target genes there is crosstalk between these pathways, CYP2B6 induction is more (Fig. 8, A–D). After cells were cultured for 48 hours post-thaw, they responsive to CAR activators compared with PXR activators (Faucette were treated with inducers for 72 hours at three concentrations prior to et al., 2006). With SC-PHH, CYP2B6 induction is generally a sensitive dmd.aspetjournals.org at ASPET Journals on September 27, 2021

Fig. 6. Metabolism-dependent cytotoxicity in cryo-HepaRG. Cryo-HepaRG were cultured for 3 days in HPRG720-supplemented media prior to initiating cytotoxicity experiments. (A, B) Aflatoxin B1 was applied to cryo-HepaRG cultures in concentration-response for 24 hours and assayed for lactate dehydrogenase leakage (A) (CytoTox-One; Promega) and ATP content (B) (CellTiter-GLO; Promega) in the presence/absence of ketoconazole (20 mM). Data represent the mean and standard deviation of three independent replicates for each treatment group. (C) Concentration-response of aflatoxin B1 in cryo-HepaRG cultures (7 days in culture) using HPRG720 (metabolism media) and HPRG770-supplemented media after 24 hours of treatment with aflatoxin B1. Data represent mean of three independent replicate wells, and error bars represent the standard deviations of the respective mean responses. Hepatocyte Function of Cryo-HepaRG Cultures 1473

150

100

50

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Fig. 7. Active uptake transport in cryo-HepaRG. Cryo-HepaRG were cultured for 10 days in HPRG720-, HPRG740-, and HPRG750-supplemented media and subsequently evaluated for active uptake transport with taurocholate and estrone sulfate. (A) Concentration-response curves for taurocholate uptake at 37°Cand4°C in cryo-HepaRG cultures. (B) Representative concentration-response curves for taurocholate uptake at 37°Cand4°C in SC-PHH (Hu8083). (C) Box and whisker plot of taurocholate uptake distributions (at 37°C) for 76 donor preparations of sandwich-cultured primary human hepatocytes (black) and day-10 cultures of cryo-HepaRG (red diamond). (D, E) Estrone sulfate uptake in cryo-HepaRG in culture for 10 days in metabolism media (HPRG720), serum-containing induction media (HPRG740), and serum-free induction media (HPRG750) media supplements (cryo-HepaRG were initially plated in HPRG770-plating/maintenance media and changed ;24 hours postplating) and assayed for estrone sulfate at 0.5 and 40 mM concentrations. Data represent mean of three or four independent replicate wells, and error bars represent the standard deviations of the respectivemeanresponses. marker of hepatocyte culture integrity, with lower quality lots failing to manner with both direct (CITCO) and indirect (PB) activators of CAR at support CYP2B6 induction in response to PB (unpublished observations sufficiently low concentrations for observing induction and minimizing over hundreds of donor preparations of SC-PHH). In cryo-HepaRG the contributions of PXR to CYP2B6 induction (Fig. 8B). Induction of cultures, CYP2B6 was clearly inducible in a concentration-related CYP2B6 in response to indirect activators (i.e., PB and phenytoin) has

TABLE 4 Uptake OATP1B1/1B3 transport with cryo-HepaRG and overlay effects

Mean ES Uptake Mean ES Uptake Treatment Group Mean E -17bG Uptake 6 S.D. (37°C) Mean E2-17bG Uptake 6 S.D. (4°C) 6 S.D. (37°C) 6 S.D. (4°C) 2

pmol/mg-min pmol/mg-min pmol/mg-min pmol/mg-min 0.5 mM HPRG720 overlay 16.9 6 0.689 3.38 6 0.363 1.33 6 0.161 0.348 6 0.127 0.5 mM HPRG720 15.3 6 0.323 3.74 6 0.220 0.701 6 0.273 0.303 6 0.0204 40 mM HPRG720 overlay 226 6 13.8 54.0 6 5.52 33.1 6 8.31 11.6 6 7.32 40 mM HPRG720 222 6 21.0 70.8 6 7.65 22.2 6 7.21 11.0 6 5.35 0.5 mM HPRG740 overlay 11.0 6 1.81 2.92 6 0.512 0.602 6 0.0489 0.154 6 0.0418 0.5 mM HPRG740 7.46 6 0.112 1.98 6 0.276 0.356 6 0.0219 0.185 6 0.0181 40 mM HPRG740 overlay 111 6 23.8 31.7 6 3.09 17.8 6 1.82 7.07 6 1.45 40 mM HPRG740 91.1 6 4.47 37.7 6 8.63 13.7 6 0.889 6.02 6 1.07 0.5 mM HPRG750 overlay 14.8 6 0.892 1.95 6 0.380 0.438 6 0.0707 0.181 6 0.0390 0.5 mM HPRG750 8.48 6 0.317 1.68 6 0.126 0.357 6 0.0181 0.244 6 0.0229 40 mM HPRG750 overlay 183 6 7.04 48.0 6 16.2 16.1 6 1.87 8.32 6 1.34 40mM HPRG750 129 6 3.33 38.2 6 7.45 17.5 6 2.29 6.50 6 1.63 1474 Jackson et al. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 27, 2021

Fig. 8. P450 enzyme induction as a marker of hepatocyte functionality with cryo-HepaRG. Cultures of cryo-HepaRG were maintained in serum-free induction media (HPRG750) for ;24 hours prior to initiating induction experiments with prototypical receptor activators for AhR, CAR, PXR, and FXR. Receptor activators were applied at three concentrations to monitor concentration-responsiveness and were applied at “lower” concentrations less prone to crosstalk with other receptor pathways (e.g., CAR and PXR with PB). (A) AhR agonists b-naphthoflavone and omeprazole produced induction of CYP1A2 enzymatic activity compared with vehicle control metabolic activity indicating a functional AhR receptor pathway in cryo-HepaRG cell cultures. (B) CAR direct activator CITCO and indirect activator PB effectively induced CYP2B6 enzymatic activity at lower concentrations, suggesting a functional CAR pathway in cryo-HepaRG cell cultures. (C) PXR activators hyperforin and rifampicin (RIF) were effective in inducing CYP3A4/5 enzymatic activity consistent with a functional PXR receptor pathway in cryo-HepaRG cell cultures. (D) ABCB11 (bile salt export pump protein) gene expression was evaluated with FXR agonist CDCA in concentration-response in cryo-HepaRG cell cultures and showed a clear induction response in both HPRG750 (serum-free) and HPRG740-supplemented induction media. (E) Summary of relationships between sentinel gene targets and hepatic receptor pathways. *Cytotoxicity was observed at 200 mM concentrations of CDCA in cryo-HepaRG cultures. Data represent the mean of three independent replicates and error bars indicate standard deviations of the mean. not been observed with any immortalized hepatic cell lines (i.e., HepG2, were effective in cryo-HepaRG, further supporting their metabolic Huh-7, Fa2N-4) to date, which has been attributed to insufficient competence akin to SC-PHH and distinguishing them from dediffer- expression and cell-signaling functionality to sequester CAR within the entiated cell lines that are generally known to have undetectable cytosol or translocate it to the nucleus (Sueyoshi et al., 2008; Templeton enzymatic activity levels. et al., 2011). CYP2B6 induction in cryo-HepaRG cultures (three Two agonists of PXR, RIF and hyperforin, were used to evaluate independent lots) was further contextualized against the range of the competence of cryo-HepaRG cultures (Fig. 8C). According to these responses observed in SC-PHH and showed comparable (and sub- data, cryo-HepaRG cultures were clearly responsive to PXR agonists stantially less variable) CYP2B6 induction in response to PB (Fig. 9). as has been reported previously with freshly differentiated HepaRG CYP2B6 mRNA induction with PB over three independent assay plates cultures (Andersson et al., 2012). Analogously to the CYP2B6 induction produced ;15-fold induction responses that were larger but consistent evaluations with cryo-HepaRG, lower concentrations of inducers with enzymatic activity induction (Fig. 10B). Consistent with the (hyperforin and RIF) were used to activate PXR at the same time CYP1A2 induction evaluations, 1-hour bupropion substrate incubations minimizing the potential for crosstalk with the CAR pathway. Induction Hepatocyte Function of Cryo-HepaRG Cultures 1475

CYP3A induction by RIF and PB was consistent with induction responses of SC-PHH in serum-free induction media. Analogously to overlay experiments with HPRG720-supplemented metabolism me- dia, the use of Geltrex overlay appeared to elevate basal levels of metabolism. In addition, the use of Geltrex overlay in general gave higher induction responses for CYP1A2, CYP2B6, and CYP3A4/5 with either HPRG740- or HPRG750-supplemented media. These data indicate that overlaying cultures of cryopreserved (and potentially freshly differentiated) HepaRG cells results in higher baseline metabolic capacities and comparable fold induction responses. The use of serum- containing or serum-free media supplements alone did not result in a substantial change in induction responsiveness with these prototypical inducers in cryo-HepaRG cultures. CAR Translocation in HepaRG Cells. To date, no immortalized hepatic cell line has been shown to effectively support the CAR signal transduction pathway, which is characterized by appreciable levels of CAR expression, cytoplasmic retention of expressed CAR, and nuclear Downloaded from translocation of CAR in response to activators such as PB. Previous Fig. 9. HepaRG and PHH induction response comparison (enzyme-specific – activities). Three separate lots of cryo-HepaRG cultures (red diamonds) were reports with overexpressed CAR green fluorescent protein fusion contextualized to ranges of induction response observed with sandwich-culture protein (transfected via expression vectors) in HepG2 cells demonstrated PHH (52 lots for CYP2B6 and CYP3A4, 11 lots for CYP1A2) for induction-of- that the CAR fusion protein spontaneously accumulates in cell nuclei, enzymatic-activity levels (box and whisker plots). P450-specific activities were resulting in constitutive activity that renders CAR nonresponsive to its evaluated after 72 hours of treatment with 50 mM omeprazole (OMP) for CYP1A2 (phenacetin O-deethylation activity), 1 mM PB for CYP2B6 (bupropion hydroxylase activators like PB, which rely on cytosolic sequestration and nuclear activity), or 10 mM RIF for CYP3A4/5 (midazolam 1-hydroxylation and testosterone translocation as an activation mechanism (Kobayashi et al., 2003). dmd.aspetjournals.org 6b-hydroxylation activities). Fold over control values were calculated relative to Proper CAR sequestration and translocation are hallmark features respective vehicle controls. Statistical analysis of lot variation with cryo-HepaRG was performed using an analysis of variance followed by a Tukey’s pairwise comparison of hepatocyte functionality that has only been shown in vivo or with revealed no statistically significantly differences at the P , 0.05 level as shown in cultures of PHH to date. To evaluate the function of the CAR Table 5. cytoplasmic retention mechanism within cryo-HepaRG, cells were thawed, plated, and infected with adenovirus-expressing EYFP-hCAR. Cells were cultured in serum-containing and serum-free media condi- of CYP3A4/5 metabolic activity compared favorably with the range of tions treated with vehicle or with 1 mM PB. Fluorescent images were at ASPET Journals on September 27, 2021 responses observed with SC-PHH (Fig. 9). The inter-lot variation with captured ;8 hours after treatment to examine the cellular localization of cryo-HepaRG (three independent lots show no statistically significant EYFP-hCAR within HepaRG cells (Fig. 12). Fluorescent images and differences, Table 5) was profoundly lower than the variability observed cell counts demonstrated that EYFP-hCAR was predominantly localized across 52 SC-PHH preparations. CYP3A4 mRNA was also effectively to the cytoplasm of HepaRG cells (70% cytosolic, 15% nuclear, and induced with 10 mM RIF over three independent assay plates. The 15% mixed) without activation. In contrast, over 90% of EYFP-hCAR ;60-fold mRNA induction produced was larger than the fold changes accumulated in the nuclei of PB-treated HepaRG cells. For the first observed at the enzymatic activity level (Fig. 10C). time in a hepatic cell line, these data demonstrate the existence of a The farnesoid X receptor is an important regulator of bile homeosta- functioning CAR cytoplasmic retention mechanism, as well as the sis, and plays an important role in liver function (Teodoro et al., 2011). ability of CAR to translocate to the nucleus in response to activation by We assessed FXR functionality in cryo-HepaRG cultures with the PB in cryo-HepaRG. agonist chenodeoxycholic acid (CDCA) and measured induction of ABCB11 mRNA expression (Fig. 8D). ABCB11 is a FXR target gene encoding for the bile salt export pump, which is heavily involved in bile Discussion acid homeostasis and plays an important role in biliary efflux of bile More than 300 publications have reported studies with HepaRG cells, acids that can be quantitatively modeled in vitro with SC-PHH (Plass with the majority of research focused on freshly differentiated HepaRG et al., 2002),(Liu et al., 1999). With CDCA treatment we observed a rather than the newer cryopreserved format (cryo-HepaRG). Cryo- concentration-related induction of ABCB11 in cultures of cryo-HepaRG HepaRG, although significantly more accessible and experimentally consistent with reports in SC-PHH (Yu et al., 2002). These data suggest flexible, differ from freshly differentiated HepaRG with respect to their that HepaRG cells support the FXR signaling pathway. detachment from freshly differentiated monolayers (disrupting cell-cell/ We further explored the effects of cell culture media supplements and cell-matrix interactions important for hepatocyte function), cryopreser- Geltrex overlay with cryo-HepaRG on liver enzyme inducibility (Fig. 11). vation, thawing, and reattachment. Although it has been claimed that Here we compared CYP1A2, CYP2B6, and CYP3A4/5 enzymatic HepaRG cell cultures (fresh and cryopreserved) are comparable to induction responses with both the HPRG740 (serum-containing) and SC-PHH, comparisons have been limited in functional coverage and HPRG750 (serum-free)-supplemented media in the presence and broader contextualization to ranges of interindividual variation. In this absence of Geltrex overlay. Cultures were treated for 72 hours with report we address these limitations and identify an initial adaptation prototypical AhR, CAR, and PXR activators. In general, comparable period in which cryo-HepaRG recover functionality. We provide fold induction responses were observed with either HPRG740- or broader contextualization of cryo-HepaRG metabolic competence, HPRG750-supplemented media using prototypical inducers in the uptake transport, and liver enzyme inducibility to ranges of interindi- presence or absence of Geltrex overlay. However, with overlay, serum- vidual variation observed with PHH, as well as genotyping analysis of free HPRG750-supplemented media supported more robust induction pharmacologically important drug clearance alleles. To our knowledge, responses for CYP1A2 and CYP2B6, whereas the more balanced this is the first report of the effect that extracellular matrix overlay has on 1476 Jackson et al. Downloaded from dmd.aspetjournals.org

Fig. 10. P450 mRNA induction of CYP1A2, CYP2B6, and CYP3A4/5 with cryo-HepaRG cultures. Cryo-HepaRG cultures were treated with omeprazole (OMP) at 50 mM, PB at 1 mM, and RIF (RIF) at 10 mM for 48 hours, and mRNA expression was quantified via quantitative reverse transcription-polymerase chain reaction (qRT-PCR) using at ASPET Journals on September 27, 2021 gene-specific TaqMan assays. (A) CYP1A2 mRNA induction in three independent assay plates. (B) CYP2B6 mRNA induction in three independent assay plates. (C) CYP3A4 mRNA induction in three independent assay plates. Data represent mean responses of three independent replicate wells and error bars indicate standard deviations of mean responses. cryo-HepaRG drug metabolism, transport, and liver enzyme induction. to the loss of cell-cell interactions and cell-matrix interactions shifting Finally, we demonstrate for the first time that cryo-HepaRG can these epithelial cells to more proliferative states. A similar process sequester CAR in the cytosol of hepatocytes and translocate CAR to was observed with the cryo-HepaRG. Freshly differentiated HepaRG hepatocyte nuclei upon PB treatment, analogously to CAR disposition in cultures disrupted during detachment/cryopreservation appeared to re- liver and PHH. quire a form of reorganization into organized, differentiated “epithelia” It is well known that hepatocyte cell-cell interactions are key drivers of upon reattachment. However, unlike PHH, cryo-HepaRG were able to hepatocyte functionality with PHH that are disrupted during cell isolations recover their fresh-like state for multiple weeks in culture. Both freshly from liver (Hamilton et al., 2001). This can result in dedifferentiation differentiated HepaRG and cryo-HepaRG appeared to form two distinct and reduced drug-metabolizing enzyme expression/activity, attributed cell types with “cords” of hepatocyte-like cells. These networks did not immediately reform but adaptively matured over 3–4 days, stabilizing by ;7–10 days to fresh-like topologies that persisted for at least 22 days. Although the mechanisms of how cryo-HepaRG cultures TABLE 5 recover fresh-like topologies are not clear, possible mechanisms might Inter-lot reproducibility include migration of hepatocyte-like cells in culture or some form of ANOVA cellular transdifferentiation (Cerec et al., 2007) whereby hepatocyte- Metabolism-Specific Activity Tukey’s (HSD) Comparison Significance P Value like and cholangiocyte-like cells switch identities. After ;10 days of CYP1A2 (APAP) 0.212 L1-L2 No recovery/redifferentiation, enzymatic activities with cryo-HepaRG were L2-L3 No restored to levels comparable to cryo-HepaRG suspension activities that L1-L3 No were substantially higher (;7-fold on average) than day-4 activities CYP2B6 (OHBP) 0.692 L1-L2 No L2-L3 No across the enzyme panel. Since drug-metabolizing enzymes are L1-L3 No established markers of hepatocyte differentiation, lower day-4 metabolic CYP3A4/5 (OHMDZ) 0.124 L1-L2 No competence, coupled with cell-morphology observations, indicates an L2-L3 No L1-L3 No initial loss of cellular differentiation with cryo-HepaRG. A more detailed CYP3A4/5 (6bT) 0.247 L1-L2 No look at the temporal kinetics of CYP3A4/5 activity loss during the initial L2-L3 No 72 hours revealed a “half-life” of ;34 hours with cryo-HepaRG cultures L1-L3 No (;10-fold decrease) that is analogous to the initial loss of metabolic ANOVA, analysis of variance; HSD, honest significant difference. competence in PHH (;1–3 hours half-life in suspensions, ;20–30 hours Hepatocyte Function of Cryo-HepaRG Cultures 1477 Downloaded from dmd.aspetjournals.org

Fig. 11. Impact of media and overlay (Geltrex) on induction with cultures of cryo-HepaRG. In these experiments the effects of Geltrex overlay in cryo-HepaRG cultures on induction responses were evaluated by assaying enzymatic activities in response to specific receptor activators for AhR, CAR, and PXR and by monitoring CYP1A2 (A), CYP2B6 (B), and CYP3A4/5 (C) enzymatic activities after 72 hours of exposure. Data represent the mean of three independent wells with error bars showing the standard deviations of mean responses. Asterisk (*) indicates P , 0.05 (Tukey’s pairwise comparison) relative to respective DMSO control groups for each culture media/overlay at ASPET Journals on September 27, 2021 combination.

in cultures) (Smith et al., 2012). However, PHH cultures do not recover (Supplemental Fig. 2). Additionally, we observed disproportionately their respective suspension enzyme activity levels under standard higher levels of certain P450s across the panel of enzymes over time, conditions (serum-free, no DMSO); instead, activity levels stabilize after suggesting a simple proliferation mechanism was not responsible solely the initial loss of metabolic competency to ;10% of their suspension for the recovery of metabolic competence. This also suggests that levels as they adapt to their new culture environment, which lasts until metabolic competence with cryo-HepaRG was not a static, intrinsic they begin to reach the end of their longevity (typically ;1week). property. The disproportionately high levels of CYP2B6, CYP3A4/5, Presumably, this lack of recovery in PHH is the result of the limitations of and UGT with cryo-HepaRG (day-10) were seemingly superphysiologic standard in vitro culture models that lack tissue-like three-dimensionality compared with activities in SC-PHH. However, these levels were lower and dynamic flow (Godoy et al., 2013). In the case of cryo-HepaRG, than median suspension PHH levels (Fig. 4). A probable explanation for the use of high concentrations of DMSO in metabolism/differentiation these disproportionate levels of inducible enzymes is a combination of media probably contributes to the observed differences. hepatocyte redifferentiation and induction by the cell culture media that In the broader context, metabolic competence with both SC-PHH mimics constitutively induced Zone 3 of a liver lobule. Previous and cryo-HepaRG was lower than suspension PHH levels, which may reports have shown that higher levels of DMSO can induce CYP3A4 limit the ability of these models to generate physiologically relevant (Nishimura et al., 2003). Further research to explore the consequences of metabolites or to model metabolic clearance rates. However, these disproportionate metabolic competence on metabolite profiles is models do effectively model liver enzyme induction. In vivo, liver warranted. enzymes are often zonally enriched, with higher constitutive levels in Overlay of cryo-HepaRG cultures with Geltrex, an extracellular Zone 3 and lower, more inducible levels in Zone 2. With cryo-HepaRG, matrix commonly used to improve longevity and quality with PHH, we observed a similar phenomenon with varied media supplementation yielded a surprising effect on cryo-HepaRG morphologies. We observed that may reflect “zonal” modeling. It is not clear how this “zonality” with higher apparent hepatocyte-to-cholangiocyte ratios along with corre- cryo-HepaRG relates to changes observed during their readaptation sponding elevations in basal metabolic competence (;2- to 3-fold). This period. “sandwich” culture approach is not required with HepaRG cells, but the One possible explanation for the recovery of metabolic competence high concentrations of DMSO that are used to differentiate HepaRG with cryo-HepaRG is that cellular proliferation might be occurring. This cells (Gripon et al., 2002) can also inhibit some P450 enzymes (e.g., is improbable, however, because we observed no evidence of increasing CYP2E1). CYP2E1 is involved in many examples of metabolically cell numbers and HepaRG are thought to be contact-inhibited (Gripon activated toxicity (i.e., acetaminophen), and overlay of cryo-HepaRG et al., 2002). Total protein levels confirmed a lack of evidence for may support more mature differentiation and require lower DMSO profoundly increased cell numbers over time with cryo-HepaRG levels (Easterbrook et al., 2001; Trafalis et al., 2010). Recent reports 1478 Jackson et al.

SC-PHH. One notable difference with cryo-HepaRG cultures was the reduced magnitude of CYP2B6 induction in response to rifampicin, and further research to explore this mechanism is warranted. Geltrex overlay had little if any effect on the fold induction of liver enzyme levels or the muted CYP2B6 rifampicin response but did elevate basal levels of metabolic capacity. CAR activators PB (indirect) and CITCO produced induction responses suggestive of a highly functional CAR pathway in cryo-HepaRG. Further exploration of this mechanism confirmed that cryo-HepaRG cultures support nuclear sequestering of CAR in the cytosol and translocation to hepatocyte nuclei upon PB treatment. CAR translocation was not observed in the cholangiocyte-like cells but was observed with and without serum. In conclusion, this report provides extensive evidence demonstrating the functionality of cryo-HepaRG and contextualizes various hepatocyte functionalities to ranges of response observed with SC-PHH. The work identifies important temporal dynamics, reveals the beneficial effects of extracellular matrix overlay, provides important genotyping data, and Downloaded from provides the first evidence for a functional CAR translocation pathway with cryo-HepaRG.

Acknowledgments The authors thank Manda Edwards and Ashley Ganoe for laboratory support with overlay experiments. The authors also thank Patricia (Miki) Pawlowski dmd.aspetjournals.org and Jasminder Sahi for contributions to reference primary hepatocyte uptake transport data; and acknowledge Alex Merrick, Sreenivasa Ramaiahgari, Bryan Fig. 12. CAR translocation in cryo-HepaRG cultures in response to phenobarbital. Mackowiak, Jessica Bonzo, Rafal Witek, and Mark Powers for the efforts in the Cryo-HepaRG cultures were plated in HPRG770-supplemented media and allowed preparation of this manuscript. to equilibrate overnight for ;24 hours prior to changing media to HPRG750 (A and B) or HPRG740 (C and D) induction media. Cryo-HepaRG were transduced with a Authorship Contributions green fluorescent protein–tagged CAR using an adenoviral delivery system and treated with indirect CAR activator PB or vehicle control (0.1% DMSO). CAR Participated in research design: Jackson, Li, Chamberlain, Wang, Ferguson. translocation was evaluated using a fluorescent microscope after 8 hours of Conducted experiments: Jackson, Li, Chamberlain, Ferguson. at ASPET Journals on September 27, 2021 treatment with PB, revealing a clear translocation of CAR in both serum-free and Performed data analysis: Jackson, Li, Wang, Ferguson. serum-containing induction media. Wrote or contributed to the writing of the manuscript: Jackson, Li, Chamberlain, Wang, Ferguson.

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Authors: Jonathan P. Jackson, Linhao Li, Erica Deibert, Hongbing Wang, and Stephen S. Ferguson

Journal Title: Drug Metabolism & Disposition

Supplemental Figure 1: Plating Cell Density Evaluation with Cryo-HepaRG. Cryo-HepaRG plating

(seeding) cell densities (75,000, 100,000, and 150,000) were produced and evaluated via cell

morphology observations and enzymatic activity measurements for CYP1A2 (phenacetin O-

deethylase), CYP2B6 (bupropion hydroxylase), and CYP3A4/5 (testosterone 6ß-hydroxylase)

enzymatic activities. Data represent the mean response of 3 independent wells and error bars reflect

standard deviations of the mean responses. Asterisk (*) indicates statistically significant (P<0.05)

using Tukey’s pairwise comparison to 75,000 cells per well response.

Supplemental Figure 2: Total protein Assays with Cryo-HepaRG Cultures over Time to Evaluate

Potential Cellular Proliferation. Due to the observed increases in enzymatic activity over time in culture with Cryo-HepaRG, total protein assays were employed after 4, 10, and 21 days in culture to assess the hypothesis that increased metabolic competence over time can be accounted for via cellular proliferation. Data represent mean responses of 3 independent well and error bars indicate standard deviations of mean responses. No statistically significant differences were observed using

Tukey’s pairwise comparison to the Day-4 control.