JPET #195750

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Evaluation of OATP1B1 and CYP3A4 activities in primary human

hepatocytes and HepaRG cells cultured in a dynamic three-

dimensional bioreactor system

Maria Ulvestad, Malin Darnell, Espen Molden, Ewa Ellis, Anders Åsberg, and Tommy B.

Andersson

DMPK Innovative Medicines, AstraZeneca R&D Mölndal, Mölndal, Sweden (M.U.; M.D.;

T.B.A.); Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo,

Oslo, Norway (M.U.; E.M.; A.Å.); Cellectis Stem Cells, Cellartis AB, Gothenburg, Sweden

(M.U.); Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm,

Sweden (M.D.; T.B.A.); Department of Clinical Science, Intervention and Technology,

Karolinska Institutet, Stockholm, Sweden (E.E.)

JPET Fast Forward. Published on July 12, 2012 as DOI:10.1124/jpet.112.195750

Copyright 2012 by the American Society for Pharmacology and Experimental Therapeutics.

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Running title: OATP1B1 and CYP4A4 activities in 3D hepatocyte bioreactors

Corresponding author:

Dr. Tommy B. Andersson

DMPK Innovative Medicines

AstraZeneca R&D Mölndal

Pepparedsleden 1

SE-431 83 Mölndal, Sweden

Phone: +46 31 776 15 34

Fax: +46 31 776 37 60

E-mail address: tommy.b.andersson@astrazeneca.com

Number of text pages: 44

Number of tables: 5

Number of figures: 9

Number of references: 58

Number of words in the abstract: 238

Number of words in the introduction: 576 (720 included references)

Number of words in the discussion: 1472 (1627 included references)

Abbreviations: OATP, organic anion-transporting polypeptide; 2D, two-dimensional; 3D,

three-dimensional; CYP, cytochrome P450; UGT, UDP-glucuronosyltransferase; E17βG,

estradiol-17β-D-glucuronide; ATA, atorvastatin acid; ATL, atorvastatin lactone; E3S, estrone-

3-sulfate; ACN, acetonitrile; LC/MS, liquid chromatography/mass spectrometry; PCR,

polymerase chain reaction; Ct, cycle threshold; ALAT, alanine aminotransferase; ASAT,

aspartate aminotransferase; ALP, alkaline phosphatise; GGT, gamma-glutamyl transferase;

MRP, multidrug resistance-associated protein; MDR, multidrug resistance protein; BCRP,

breast cancer resistance protein.

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Abstract

Long-term stability of liver cell functions is a major challenge when studying hepatic drug

transport, metabolism and toxicity in vitro. The aim of the present study was to investigate

the OATP1B1 and CYP3A4 activities in fresh primary human hepatocytes and differentiated

cryopreserved HepaRG cells when cultured in a 3D bioreactor system. The OATP1B1

activity was determined by loss from media experiments of [3H]-estradiol-17β-D-glucuronide

(3H-E17βG) and atorvastatin acid (ATA) for up to 7 days in culture. ATA metabolite formation

was determined at day 3 to 4 to evaluate the CYP3A4 activity. Overall, the results showed

that freshly isolated human hepatocytes inoculated in the bioreactor retained OATP1B1

activity for at least 7 days, while in HepaRG cells, no OATP1B1 activity was observed

beyond day 2. The activity data were in agreement with immunohistochemical stainings,

which showed that OATP1B1 protein expression was preserved for at least 9 days in fresh

human hepatocytes, while OATP1B1 was markedly lower expressed in HepaRG cells after 9

days in culture. Fresh human hepatocytes and HepaRG cells exhibited similar CYP3A4

activity in bioreactor culture, and immunohistochemical stainings supported these findings.

Activity and mRNA expression of OATP1B1 and CYP3A4 in primary human hepatocytes

compared to HepaRG cells in fresh suspensions were in agreement with data obtained in

bioreactor culture. In conclusion, freshly isolated human hepatocytes cultured in a 3D

bioreactor system, preserves both OATP1B1 and CYP3A4 activities, allowing long-term in

vitro studies on drug disposition and toxicity.

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Introduction

Drug-drug interactions, large inter-patient variability in pharmacokinetics, and the propensity

of drugs to induce liver injury, are major concerns when developing new drugs. Hepatic

membrane transporters are today recognized as important determinants of disposition and

safety of many drugs, and work in cooperation with metabolizing enzymes in order to restrict

systemic exposure of exogenous substances (International Transporter Consortium et al.,

2010). Several transporter-mediated drug-drug interactions have been reported (Zhang et al.,

2009), and inhibition of hepatic uptake and/or efflux has been indicated as a possible

mechanism of hepatotoxicity (Kostrubsky et al., 2003; Marion et al., 2007; Morgan et al.,

2010; Ye et al., 2010). Moreover, hepatic membrane transporters are major determinants of

pharmacokinetic variability of many drugs (International Transporter Consortium et al., 2010).

In drug development, it is therefore crucial to develop in vitro models for drug disposition that

can be run long enough to detect the above mentioned effects at clinically relevant

concentrations. These models should also express activities of membrane transporters and

drug-metabolizing enzymes reflecting the in vivo situation.

While cytochrome P450 3A4 (CYP3A4) has been identified as the most frequently involved

enzyme in drug metabolism (Guengerich, 1999), the organic anion-transporting polypeptide

1B1 (OATP1B1) is crucial for uptake of many acidic drugs into the liver, e.g. HMG-CoA

reductase inhibitors (statins) (Hirano et al., 2004; Nakai et al., 2001). Several OATP1B1-

mediated drug-drug interactions involving these agents have been reported (Lau et al., 2007;

Nakagomi-Hagihara et al., 2007; Noe et al., 2007; Shitara and Sugiyama, 2006; Treiber et

al., 2007). An important example is the substantially increased statin exposure and risk of

toxicity observed during co-administration with the potent OATP1B1 inhibitor cyclosporine A

(Amundsen et al., 2010; Asberg et al., 2001).

Primary hepatocytes, plated or in suspension, represent the most common in vitro model for

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evaluation of human hepatic drug transport, metabolism, clearance and toxicity in the

pharmaceutical industry (Hewitt et al., 2007). These cells express a complete set of enzymes

and transporters involved in hepatic drug clearance, but their spontaneous dedifferentiation

and rapid loss of enzyme and transporter activity in two-dimensional (2D) culture prevents

long-term studies (Richert et al., 2006; Rodriguez-Antona et al., 2002; Ulvestad et al., 2011).

Furthermore, restricted tissue availability and inter-donor variability due to genetic differences

and variability in the cell preparation steps limits the use of primary human hepatocytes (Li,

2008). This has led to the development of protocols for cryopreservation of human

hepatocytes. Cryopreserved human hepatocytes exhibit both cytochrome P450 (CYP), UDP

glucuronosyltransferase (UGT) and transporter activities (Badolo et al., 2011; De Bruyn et

al., 2011; Fournel-Gigleux et al., 2010; Jorgensen et al., 2007; Li et al., 1999; Steinberg et

al., 1999), but as in primary human hepatocytes, a rapid loss of enzyme and transporter

expression is observed when these cells are cultured in 2D models (Richert et al., 2006).

Thus, novel cell culture systems with improved conservation of hepatocyte functions allowing

predictive long-term in vitro pharmacokinetic or toxicological studies are warranted.

In 1994, Gerlach et al. introduced the multicompartment bioreactor technology for dynamic

three-dimensional (3D) perfusion culture of human liver cells (1994). This technique uses

interwoven hollow-fiber capillary membranes that provide independent, decentralized

medium and gas exchange to the cells located between the capillaries (Fig 1B). When

cultured in a perfused 3D bioreactor, human liver cells retain in vivo-like properties and are

arranged in tissue-like structures (Schmelzer et al., 2009; Zeilinger et al., 2004; Zeilinger et

al., 2004). Zeilinger et al. (2002) showed that liver-specific functions such as urea and

albumin synthesis, glucose metabolism and CYP activities were all maintained for at least 14

days in bioreactor culture.

HepaRG cells, a human hepatoma cell line, represents an alternative to primary human

hepatocytes. Differentiated HepaRG cells maintain important functions for drug metabolism,

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such as CYP and UGT activities, both in 2D and 3D models over several weeks (Antherieu et

al., 2010; Darnell et al., 2011; Josse et al., 2008). Furthermore, Le Vee et al (2006) reported

activity of certain canalicular and biliary transporters in HepaRG cells in 2D culture.

Preservation of drug transport and metabolism activities over longer time periods is essential

in in vitro liver models for drug discovery research. The aim of the present study was to

investigate the OATP1B1 and CYP3A4 activities in fresh primary human hepatocytes and

differentiated cryopreserved HepaRG cells cultured in a 3D bioreactor system over several

days.

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Materials and Methods

Chemicals and reagents. [3H]-E17βG was obtained from Perkin Elmer (Boston, MA, USA).

ATA, atorvastatin lactone (ATL), 2-OH-atorvastatin acid (2-OH-ATA), 2-OH-atorvastatin

lactone (2-OH-ATL), 4-OH-atorvastatin acid (4-OH-ATA) and 4-OH-atorvastatin lactone (4-

OH-ATL) were obtained from Toronto Research Chemicals (Toronto, Canada). Unlabeled

estradiol-17β-D-glucuronide, estrone-3-sulfate (E3S) and L-glutamine (200 mM) were

obtained from Sigma-Aldrich (Steinheim, Germany). Cryopreserved human hepaticytes were

obtained from Celsis In Vitro Technologies (Brussels, Belgium). Differentiated cryopreserved

HepaRG cells were obtained from Biopredic International (Rennes, France). HEPES,

phosphate-buffered saline and Williams’ Medium E without phenol red were obtained from

Invitrogen (Carlsbad, CA, USA). HCl and NaOH were obtained from Merck (Darmstadt,

Germany). High-purity water was obtained from an ELGA purification system (ELGA, High

Wycombe, UK). All other chemicals and reagents were of analytical grade and were

available from commercial sources. Bioreactors, tubing systems and perfusion device were

purchased from Stem Cell Systems (Berlin, Germany).

Isolation of human hepatocytes. All tissues were obtained from Karolinska University

Hospital (Huddinge, Sweden) by qualified medical staff, with donor informed consent

following ethical and institutional guidelines. Liver tissues from three patients (donor 1, 2 and

3) undergoing liver resection due to metastatic colon or liver cancer were used in this study.

Donor information regarding age, sex, disease and cell viability is summarized in Table 1.

Residual tissue not needed for diagnostic purposes was transported to the laboratory from

the operating rooms in cold Eagle’s Minimum Essential Medium (EMEM) (Lonza, Walkerville,

MD) within 30 min after removal. Tissue dissociation and hepatocyte isolation were

performed using a two-step collagenase perfusion procedure originally developed by Seglen

(1976) and modified as described in detail by Gramignoli et al. (2011). The isolated fresh

human hepatocytes were transported from Karolinska University Hospital, Huddinge, to

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AstraZeneca R&D Mölndal in a cold package within the same day. Immediately upon arrival,

the cells were centrifuged for 3 min at 100 g and the supernatant was removed. The cells

were washed with 45 mL Williams Medium E without phenol red supplemented with 25 mM

HEPES and 2 mM L-glutamine, pH adjusted to 7.4 (modified Williams Medium E) and

centrifuged for 3 min at 100 g. The cells were counted using a Bürker chamber and trypan

blue exclusion test was used to calculate the cell viability. The cell suspension was diluted in

medium to required concentration. The cells were kept on ice and used within 3 h after

isolation.

Thawing of cryopreserved human hepatocytes and HepaRG cells. Cryopreservation of

human hepatocytes was conducted at Celsis In Vitro Technologies (Brussels, Belgium) using

a controlled freezing protocol and according to in-house procedures. Pooled cryopreserved

human hepatocytes from three different batches (IRK, UMJ and PHL), each containing

hepatocytes from ten donors, were used in the experiments. Immediately before experiments

in suspension, cryopreserved human hepatocytes were rapidly thawed by gently shaking the

vial in a 37 °C water bath. The content was transferred to a 50 mL tube containing pre-

warmed (37 °C) modified Williams Medium E. The cryogenic tubes were rinsed twice and the

50 mL tube was centrifuged at 60 g for 5 min. After removal of the supernatant, the pellet

was resuspended in 2-3 mL buffer.

HepaRG cells were differentiated and cryopreserved at Biopredic International (Rennes,

France), and three different batches (HPR116046, HPR116048 and HPR116070) of

cryopreserved HepaRG cells were used in the experiments. Immediately before experiments

in suspension or inoculation of cells into the bioreactor, HepaRG cells were rapidly thawed

by gently shaking the vial in a 37 °C water bath. The content was transferred to a 50 mL tube

containing pre-warmed (37 °C) basal hepatic cell medium MIL600 supplemented with

ADD670 according to the manufacturer’s instructions (Biopredic International). The cryogenic

tubes were rinsed twice. The 50 mL tube was centrifuged at 360 g for 2 min. After removal of

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the supernatant, the pellet was resuspended in 2-3 mL basal hepatic cell medium MIL600

supplemented with serum-free ADD650 according to the manufacturer’s instructions

(Biopredic International).

Cryopreserved human hepatocytes and HepaRG cells were counted using a Bürker chamber

and trypan blue exclusion test was used to calculate the cell viability. The cell suspensions

were diluted with respective buffer to required concentration. The cells were kept on ice and

used within 3 h after thawing.

Bioreactor and perfusion system. The bioreactor consists of two hollow fiber capillary

layers integrated into a polyurethane housing. Each capillary layer includes hydrophobic

oxygenation capillaries and hydrophilic medium perfusion capillaries (Fig. 1B). The medium

capillaries were arranged as two layers with counter-current perfusion in order to increase

mass exchange between the inside of capillaries and the cells located within the inter-

capillary space. A port enabled substance injection, mimicking bolus administration, and

sampling from the re-circulating medium (Fig. 1A). Fresh medium was continuously supplied

from the medium bottle and excess medium flowed into the outflow bottle (Fig. 1A). A

detailed description of the bioreactor structure is given elsewhere (Hirano et al., 2006). In this

study, a down-scaled bioreactor with a cell compartment volume of 0.5 mL was used for

comparison with the 2 ml cell compartment bioreactor used in previous studies (Darnell et al.,

2011, Zeilinger et al, 2011). The bioreactors were operated by means of an electronically

controlled perfusion device (Zeilinger et al, 2011) allowing two bioreactors to be run in

parallel. Temperature, medium feed and medium recirculation rates were monitored and

regulated via a connected computer. A valve-controlled gas mix unit was used for

air/oxygen/CO2 supply.

Bioreactor cell inoculation and culture. Bioreactors were connected with preassembled

sterile tubing systems and rinsed with phosphate-buffered saline and medium before cell

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inoculation. 2.5 mL cell suspension (10 x 106 viable cells/mL) was inoculated in the cell

compartment of each bioreactor to a final amount of 25 x 106 cells per bioreactor. The

bioreactors were waggled carefully during the filling to allow even distribution.

Fresh human hepatocytes from donor preparation 1, 2 and 3 were inoculated into three

bioreactors at separate occasions. The bioreactors were perfused with Heparmed medium

Vito 143 (Biochrom, Berlin, Germany), a modification of Williams’ medium E specifically

adapted for serum-free perfusion culture of liver cells by enrichment with amino acids, free

fatty acids and trace elements. Prior to use, the medium was supplemented with 20 IU/L

insulin, 5 mg/L transferrin, 3 μg/L glucagon (Biochrom), 100,000 U/L penicillin, 100 mg/L

streptomycin (Invitrogen) and 20 µg/mL gentamycin (PAA Laboratories GmbH, Pasching,

Austria). Three different batches of cryopreserved differentiated HepaRG cells were

inoculated into three bioreactors and hepatic cell medium MIL600 supplemented with

ADD670 was used during the whole culture period.

Bioreactor cultures were perfused with a total medium volume of 12 mL recirculating at a flow

rate of 3 mL/min. The temperature in the bioreactor chamber was maintained at 37°C and

bioreactors were oxygenated through the integrated gas capillaries with a gas mix of 95% air

and 5% CO2 at a gas flow velocity of 5 mL/min. Before and between experiments, medium

flow rate was set to 0.6 mL/h and the standard operation mode with continuous medium feed

(open-system perfusion) was used.

Transport studies with E17βG in cell suspension. Loss from media and intracellular

accumulation of E17βG with and without E3S (OATP1B1 inhibitor) was determined in cell

suspension from three different batches of pooled cryopreserved human hepatocytes, three

different batches of HepaRG cells and fresh human hepatocytes from donor preparation 1, 2

and 3 at different occasions. Cells were suspended in medium to a final density of 3 x 106

viable cells/mL. The experiments were performed in triplicate in 96-well plates (Nunc,

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Thermo Fisher Scientific, Roskilde, Denmark) with 50 µL cell suspension/well. The cells were

pre-incubated with either 25 µL estrone-3-sulfate (30 µM) or medium (control samples) for 10

min at 37 °C. The transport studies were initiated by adding 75 μL substrate solution giving a

final 3H-E17βG concentration of 1 µM (0.5 µCi/mL), and a final cell concentration of 1 x 106

cells/mL. The cells were incubated for 1, 4, 8, 15, 30 and 60 min. The whole sample was

transferred to an Eppendorf tube and centrifuged for 30 s at 10 000 g. 50 µL of the

supernatant was immediately transferred to a 96 well-plate (Optiplate, PerkinElmer) and 200

µL scintillation liquid (MicroScint 20, PerkinElmer) was added. Immediately after removal of

the supernatant, 350 µL ice-cold medium was added to the pellet to stop the transporter-

mediated uptake. The tubes were centrifuged for 30 seconds at 10 000 g, and the pellets

were washed twice with 350 µL ice-cold buffer. After the third centrifugation, 100 µL 1 M

NaOH was added to the pellet to lyse the cells, and the samples were placed in the fridge (4

°C) over night. 50 µL sample was transferred to a 96 well-plate (Optiplate, PerkinElmer) and

200 µL scintillation liquid (MicroScint 20, PerkinElmer) was added. The radioactivity in the

samples was determined by a microplate scintillation counter (TopCount, PerkinElmer).

Transport studies with E17βG in the bioreactor. Loss from media of 3H-E17βG with and

without E3S was assessed in fresh human hepatocytes from donor preparation 1, 2 and 3

and in two different batches of HepaRG cells at day 2 and 7 in bioreactor culture (Fig. 2). In

experiment 1, 1 mL substrate solution was injected through the sampling port (Fig. 1A) giving

a final concentration of 1 µM (0.5 µCi/mL). The transport studies were performed at

continuous medium perfusion, but without feeding medium into the perfusion circuit (closed-

system perfusion) (Fig. 1A). Samples (150 µL) were collected at 8, 20, 40, 60, 90, 120 and

180 min. The bioreactor circuit was rinsed with 50 mL culture medium (single-pass perfusion)

before experiment 2. In experiment 2, the cells were pre-incubated with E3S for 30 min by

injecting 1 mL inhibitor solution. After 30 min, 1 mL medium was removed from the

recirculation and replaced by 1 mL substrate solution giving a final concentration of 1 µM (0.5

µCi/mL) 3H-E17βG and 30 µM E3S. Except from the preincubation with inhibitor, experiment

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2 was executed exactly as experiment 1. After the experiments, the bioreactor circuit was

rinsed with 50 mL of culture medium (single-pass perfusion) and reset to the standard

operation mode with continuous medium feed (open-system perfusion). Duplicate samples of

50 µL were mixed with 5 mL scintillation liquid (OptiPhase HiSafe 2, PerkinElmer). The

radioactivity in the samples was determined by liquid scintillation spectroscopy (Wallac Win

Spectral, 1414 Liquid Scintillation Counter, PerkinElmer).

In addition to the transport studies in the bioreactor, 3H-E17βG was injected in a bioreactor

system without cells in order to assess potential non-specific binding to the bioreactor

system. Samples were analyzed at 0, 8, 20, 40, 60, 90, 120 and 180 min and the results did

not indicate any such non specific binding.

Transport and metabolism studies with ATA in cell suspension. Total loss of ATA and

ATA metabolite formation with and without E3S was determined in suspension from three

different batches of pooled cryopreserved human hepatocytes, three different batches of

HepaRG cells and fresh human hepatocytes from donor preparation 1, 2 and 3. Cells were

suspended in medium to a final density of 4 x 106 viable cells/mL. The experiments were

performed in triplicate in 96-well tissue culture plates with flat bottom (TPP, Trasadingen,

Switzerland). 50 µL cell suspension/well were pre-incubated for 10 min at 37 °C. 50 µL pre-

warmed (37 °C) substrate solution with or without E3S (final concentration 30 µM) was added

to a final concentration of 10 µM ATA, and a final cell concentration of 2 x 106 cells/mL. For

cryopreserved human hepatocytes and HepaRG cells, the plates were incubated at 37 °C for

2, 6 and 24 h. For fresh human hepatocytes, the plates were incubated at 37 °C for 2, 14 and

24 h. Longer incubations were performed with ATA compared to E17βG to ensure formation

of quantifiable levels of metabolites. The reactions were stopped with 100 µL ice-cold ACN.

The 0 h plate was stopped with 100 µL ACN before adding the substrate. The plates were

placed in the freezer (-20 °C) for 20 min and centrifuged for 20 min at 4000 g and 4 °C. The

supernatant was mixed with equal amount of ELGA water. The triplicate samples were

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pooled and stored at -80 °C until analysis. Before analysis, the samples were thawed and

mixed with equal amount of ACN. The samples were placed in the freezer (-20 °C) for 20 min

and centrifuged for 20 min at 4000 g and 4 °C. The supernatant was diluted with ELGA water

and analysed by liquid chromatography/mass spectrometry (LC/MS).

Transporter and metabolism studies with ATA in the bioreactor. Loss from media of

ATA and ATA metabolite formation with and without E3S was assessed in fresh human

hepatocytes from donor preparation 1, 2 and 3 and in three different batches of HepaRG

cells at day 3 to 4 in bioreactor culture (Fig. 2). In experiment 1, 1 mL substrate solution was

injected through the sampling port giving a final concentration of 5 µM ATA. The transport

and metabolism studies were performed in closed-system perfusion mode as described

above. Samples (150 µL) were collected at 15, 30 min, 1, 2, 4, 6 and 24 h. Longer

incubations were performed with ATA compared to E17βG to ensure formation of

quantifiable levels of metabolites. The bioreactor circuit was rinsed with 50 mL culture

medium (single-pass perfusion) before experiment 2. In experiment 2, the cells were pre-

incubated with E3S for 30 min. After 30 min, 1 mL medium was removed from the

recirculation and replaced by 1 mL substrate solution giving a final concentration of 5 µM

ATA and 30 µM E3S. Except from the pre-incubation with inhibitor, experiment 2 was

executed exactly as experiment 1. After the experiments, the bioreactor circuit was rinsed

and reset to the standard operation mode. Samples were frozen directly after sample taking.

Before analysis, the samples were thawed and mixed with ice-cold ACN (1:2). The samples

were centrifuged for 20 min at 4000 g and 4 °C. The supernatant was diluted with ELGA

water and analyzed by LC/MS.

In addition to the transport and metabolism studies in the bioreactor, ATA was injected in a

bioreactor system without cells in order to assess potential non-specific binding to the

bioreactor system. Samples were analyzed at 0, 15, 30 min, 1, 2, 4, 6 and 24 h and the

results did not indicate any such non specific binding.

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Analysis of ATA and ATA metabolites. ATA, ATL, 2-OH-ATA, 2-OH-ATL, 4-OH-ATA and

4-OH-ATL were analyzed using LC/MS. The liquid chromatography system consisted of an

HTS PAL injector (CTC Analytics, Zwingen, Switzerland) combined with an HP 1100 LC

binary pump and column oven (Agilent Technologies Deutschland, Waldbronn, Germany).

The separation was performed on a reversed-phase HyPurity C18 analytical column (50 ×

2.1 mm, 5 μm; Thermo Fisher Scientific, Waltham, MA) at 40°C and a flow rate of 0.75

mL/min. The mobile phases consisted of 0.1% (v/v) formic acid in 5% ACN (A) and 0.1%

(v/v) formic acid in 95% ACN (B). Mobile phase B was maintained at 2% for 0.8 min, and

then increased linearly from 2% to 98% for 3.7 min. After maintenance at 98% for 0.5 min,

mobile phase B was decreased linearly to 2% in 0.1 min and maintained at 2% for 0.9 min.

The retention times of 4-OH-ATA, 4-OH-ATL, 2-OH-ATA, ATA, 2-OH-ATL and ATL were

3.05, 3.20, 3.43, 3.51, 3.57 and 3.66 min, respectively. Detection was performed with a triple

quadrupole mass spectrometer, API4000, equipped with an electrospray interface (Applied

Biosystems/MDS Sciex, Concord, ON, Canada). The mass spectrometry parameters were

optimized using each analyte. The compound-dependent parameters were as follows: the

collision energy was set to 31, 28, 30, 31, 25 and 25 V for 4-OH-ATA, 4-OH-ATL, 2-OH-ATA,

ATA, 2-OH-ATL and ATL, respectively. The collision-activated dissociated gas was set to 6

for all analytes. The analytes was separated into two periods. In period 1, the multiple

reaction monitoring (MRM) transitions chosen were 575.2 > 440.3 for 4-OH-ATA and 557.1 >

448.2 for 4-OH-ATL. In period 2, the MRM transitions chosen were 575.2 > 440.2 for 2-OH-

ATA, 559.3 > 440.2 for ATA, 557.1 > 448.1 for 2-OH-ATL and 541.1 > 448.2 for ATL.

Instrument control, data acquisition, and data evaluation were performed using Analyst 1.4

software (Applied Biosystems/MDS Sciex).

Immunohistochemical staining. Samples from cell culture material were collected from

different locations within the bioreactor capillary network upon culture termination. After

fixation in 5% paraformaldehyde the material was dehydrated and embedded in paraffin and

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cut into approximately 4 µm-sections. The immunohistochemical staining was performed on

the staining module Discovery XT (Ventana® Medical Systems Inc, Tucson, USA), using

antibodies against OATP1B1 (kind gift from Dr. Bruno Stieger, University Hospital Zurich,

Zurich) and CYP3A4 (CYPEX PAP011) (Cypex Ltd, Dundee, Scotland UK). All solutions for

deparaffinization, pretreatment, detection, counterstaining and rinsing steps, were supplied

by Ventana® Medical systems Inc. Heat (40 min in a Tris/Borate/EDTA buffer pH 8, at 98°C)

were used as antigen retrieval pretreatment. Primary antibodies were diluted in Discovery Ab

Diluent. As secondary step the Ventana OmniMap anti-Rb HRP were used. The

immunohistochemical staining was visualized with diaminobenzidine chromogen (DAB) and

the counterstaining was performed with haematoxylin.

RNA isolation and cDNA synthesis. Total RNA was isolated from fresh human

hepatocytes, cryopreserved human hepatocytes and HepaRG cells using TRIzol reagent

according to manufacturers’ instructions. cDNA was prepared from 0.5 µg of total RNA using

the SuperScript ІІІ First-Strand Synthesis System for reverse transcription-polymerase chain

reaction (PCR) with random hexamer primers according to the manufacturer’s protocol

(Invitrogen, Carlsbad, CA, USA).

Quantitative real-time PCR. Quantitative real-time PCR amplification reactions were carried

out in an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster

City, CA). Gene expression was estimated using TaqMan® Gene Expression Assays with

specific primers and probes (Applied Biosystems) according to the manufacturer’s

recommendations. Briefly, the reaction mixtures (25 µL/well) contained 30 ng of cDNA, 1 X

TaqMan Universal Master Mix (Applied Biosystems, Branchburg, NJ, USA), 1.25 µL Gene

Expression Assays Mix and RNase free water. The thermal cycle had initial steps of 2 min at

50 °C for UNG activation and 10 min at 95 °C for initial denaturation of target DNA and

activation of AmpliTaq Gold DNA polymerase, followed by 40 PCR cycles of 15 s at 95 °C for

denaturation and 1 min at 60 °C for annealing and extension. Each sample was analyzed in

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duplicates and data was captured using the 7500 Real-Time PCR System Sequence

Detector Software v1.3.1 (Applied Biosystems). Gene product measured by QRT-PCR were

SLCO1B1 (Hs00272374_m1) and CYP3A4 (Hs00604506_m1). Expression levels were

normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The

relative gene expression (ΔΔCt) and the fold change of gene expression (2-∆∆Ct) were

calculated using the cryopreserved human hepatocytes as a calibrator.

Metabolic parameters and cell damage markers. Metabolic parameters and cell damage

markers in fresh human hepatocytes and HepaRG cells cultured in the 3D bioreactor were

measured daily in samples from the culture perfusate and/or the medium outflow. The

samples were stored at -20 °C and analyzed at the Laboratory of Clinical Chemistry at

Sahlgrenska University Hospital, Gothenburg. Urea and glucose production/consumption and

release of aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), alkaline

phosphatase (ALP) and gamma-glutamyl transferase (GGT) were determined in all samples.

The daily production, consumption or release of biochemical parameters in the bioreactors

was calculated on the basis of concentrations measured in the outflow bottle and in the

perfusion circuit (Fig. 1A), according to the following equation:

)( )( tVCCtVCCtP RddM Δ×−+Δ×−=Δ −100

where P is the total production rate, Δt is the time interval between two sampling time points,

CO is concentration in the outlet vessel, CM is medium blank concentration, VO is volume of

medium in the outlet vessel, Cd is concentration in perfusate at the time point of sample

taking, Cd-1 is concentration in the previously collected sample from the perfusate, and VR is

recirculating volume. The mean values and S.D.s were calculated for the three bioreactors.

The parameters are presented as milligrams per day per bioreactor or unit per day per

bioreactor.

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Calculations and statistical analysis. Non-linear regression was used to describe loss

from media data of E17βG (one phase exponential decay) and ATA (two phase exponential

decay) in bioreactor culture (Fig. 4 and Fig. 6, respectively) using GraphPad Prism 4.03

(GraphPad Software Inc., La Jolla, CA). Area under the curve (AUC) of substrate

concentration and/or metabolite concentration was determined without and with co-

incubation with E3S in each experiment using the trapezoidal method.

Triplicate experiments were performed in all studies except from in loss from media of

E17βG in HepaRG cells cultured in the bioreactor where duplicate experiments were

performed. The mean value ± S.D. were calculated. The significance of difference of AUC of

substrate concentration or metabolite concentration without and with co-incubation with E3S

was estimated using the one-tailed, paired Student’s t test. The significance of difference in

gene expression of SLCO1B1 and CYP3A4 between fresh human hepatocytes,

cryopreserved human hepatocytes and HepaRG cells was estimated using the two-tailed,

unpaired Student’s t test. P-values < 0.05 were considered as statistically significant.

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Results

Intracellular accumulation and loss from media of E17βG in cell suspension.

OATP1B1-mediated uptake of E17βG was determined by combined time-dependent uptake

studies and loss from media experiments of E17βG in cell suspensions. AUC of the E17βG

medium concentration and the intracellular E17βG concentration without and with E3S (30

µM) are shown in Table 2. Results from the time-dependent uptake studies showed an

OATP1B1-dependent active uptake of E17βG in both fresh human hepatocytes (p < 0.01),

cryopreserved human hepatocytes (p < 0.01) and HepaRG cells (p < 0.01) (Fig. 3, Table 2).

In the loss from media experiments, a significant OATP1B1-mediated loss from media of

E17βG was observed in fresh (p < 0.01) and cryopreserved (p < 0.05) human hepatocytes,

while no significant difference in loss from media of E17βG with and without E3S was

observed in the cryopreserved HepaRG cells (p > 0.05) (Table 2).

Loss from media of E17βG in cells cultured in the bioreactor system. OATP1B1-

mediated loss from media of E17βG was determined at day 2 and 7 in the bioreactor. Fig. 4

shows the E17βG medium concentration as % of start concentration without and with E3S in

fresh human hepatocytes (panel A and B) and HepaRG cells (panel C and D) at day 2 and 7,

respectively. AUC of the E17βG medium concentration without and with E3S over time are

shown in Table 3. A significant OATP1B1-mediated loss from media was observed in fresh

human hepatocytes at day 2 in culture (p < 0.05). At day 7, two out of three donors showed

an OATP1B1-mediated loss from media of E17βG (p > 0.05). In HepaRG cells, a significant

OATP1B1-mediated loss from media was observed at day 2 in culture (p < 0.05), but the

results showed no OATP1B1 activity at day 7 (p > 0.05).

Loss of ATA and ATA metabolite formation in cell suspension. Total loss of ATA and

ATA metabolite formation with and without E3S was determined in cell suspension at day 0.

The results are shown in Fig. 5. AUC of the ATA concentration and the total hydroxy

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metabolite concentration in the experiments with and without E3S are shown in Table 4.

Significantly more loss of ATA and more metabolite formation were observed in the

experiments without E3S in both fresh (p < 0.05) and cryopreserved (p < 0.05) human

hepatocytes, indicating an active OATP1B1-mediated uptake of ATA. In HepaRG cells, no

significant difference was observed in loss of ATA or metabolite formation without and with

E3S (p > 0.05). The CYP3A4 dependent formation of 2-OH-ATA acid and 4-OH-ATA were

similar in all three cell systems. Concentrations of ATL, 2-OH-ATL and 4-OH-ATL were

below the limit of quantification.

Loss from media of ATA in cells cultured in the bioreactor system. OATP1B1-mediated

loss from media of ATA was determined after 3 to 4 days in the bioreactor. Fig. 6 shows the

ATA medium concentration as % of start concentration with and without E3S in fresh human

hepatocytes and HepaRG cells. AUC of the ATA medium concentration with and without E3S

over time are shown in Table 5. Comparing the AUC values with and without co-incubation

with E3S, no statistical significant difference was observed in either fresh human hepatocytes

or HepaRG cells at day 3 to 4 in culture. However, visual examination of Fig. 6A presenting

the loss from media of ATA +/- E3S in fresh human hepatocytes, and the relatively low p-

value of 0.080 obtained when comparing AUC of ATA without and with E3S, suggests an

OATP1B1-dependent loss from media in fresh human hepatocytes.

Compared to the amount of ATA metabolites formed in the first experiment, the formation of

2-OH-ATA and 4-OH-ATA was increased in the subsequent experiment, when E3S was co-

incubated with ATA (Fig. 2), in both fresh human hepatocytes and HepaRG cells (Fig. 7), as

a result of CYP3A4 induction by atorvastatin and increased metabolite formation over time.

Concentrations of ATL, 2-OH-ATL and 4-OH-ATL were below the limit of quantification.

Immunohistochemical staining of OATP1B1 and CYP3A4. Protein expression and

cellular localization of OATP1B1 and CYP3A4 were determined in bioreactors inoculated

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with freshly isolated human hepatocytes or HepaRG cells by immunohistochemical staining

after 9 days culture. Examination of primary human hepatocytes in the bioreactor showed

that OATP1B1 was expressed and localized to the hepatocyte plasma membrane (Fig. 8,

panel A). OATP1B1 was evenly distributed on the cell surface. In HepaRG cells, the

OATP1B1 protein expression was markedly lower than in hepatocytes in bioreactor culture at

day 9 (Fig. 8, panel B). Similar protein levels of CYP3A4 were expressed in fresh human

hepatocytes and HepaRG cells (Fig. 8, panel C and D). Unfortunately, the bioreactor tissue

structure is easily ruptured due to technical difficulties in removing the fixated cell material

from the bioreactor. This phenomenon can be observed in the histological pictures of fresh

human hepatocytes in bioreactor culture, especially in panel C, but should not affect the

immunostaining of the cells.

mRNA expression of SLCO1B1 and CYP3A4. The gene expression of SLCO1B1 and

CYP3A4 in freshly isolated human hepatocytes and freshly thawed cryopreserved human

hepatocytes and HepaRG cells are shown in Fig. 9. No significant difference in the gene

expression level of SLCO1B1 was observed between fresh and cryopreserved human

hepatocytes. The SLCO1B1 gene expression level was 95% (p < 0.01) and 90% (p < 0.05)

lower in HepaRG cells than in fresh human hepatocytes and cryopreserved human

hepatocytes, respectively. No significant differences in the gene expression levels of

CYP3A4 were observed.

Metabolic parameters. Daily urea and glucose production/consumption and release of

ALAT, ASAT, ALP, GGT were determined in fresh human hepatocytes and cryopreserved

HepaRG cells cultured in the bioreactor. Daily release of ALAT, ASAT, ALP and GGT and

production/consumption of urea and glucose in fresh human hepatocytes from three different

donors cultured in separate bioreactors were similar with the exception that donor

preparation 2 showed higher ASAT and ALAT during the first two days in culture.

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Discussion

The major finding of the present study is that fresh primary human hepatocytes cultured in a

3D bioreactor system retain both OATP1B1 and CYP3A4 activities longer than in currently

available in vitro models. Activity measures were in line with conserved protein expression

during the same time period. Thus, primary human hepatocytes cultured in a 3D bioreactor

system appear to be a promising model for drug discovery research.

The rapid loss of transporter and enzyme activity in 2D cultures of human hepatocytes is a

major concern when studying drug uptake, metabolism and extrusion from cells in vitro, and

prevents reliable long-term studies to be performed (Richert et al., 2006; Rodriguez-Antona

et al., 2002; Ulvestad et al., 2011). Ulvestad et al. (2011) showed an extensive decrease in

OATP1B1/1B3 activity in plated primary human hepatocytes, along with increased passive

diffusion, when cultured for more than 2 hours. In the bioreactor inoculated with freshly

isolated human hepatocytes, OATP1B1 activity was retained for at least 7 days, while in

HepaRG cells, no OATP1B1 activity was observed later than day 2. The activity data were in

agreement with immunohistochemical stainings which showed that OATP1B1 protein

expression was preserved for at least 9 days in fresh human hepatocytes, while in HepaRG

cells, OATP1B1 was markedly lower than in hepatocytes at day 9 in culture, indicating that

freshly isolated human hepatocytes is preferred compared to HepaRG cells. Hoffmaster et al.

(2004) have previously reported that the OATP1B1 and OATP1B3 protein expression is

retained in sandwich-cultured human hepatocytes for up to six days, but the metabolic

capacity of this model has been questioned.

A significant OATP1B1-mediated uptake of E17βG was observed in all three cell types in

suspension. However, the OATP1B1-mediated transport was significantly higher in human

hepatocytes than in HepaRG cells, both in suspension and in bioreactor culture at day 2. The

OATP1B1 activity data are consistent with the mRNA expression of SLCO1B1 in cell

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suspensions showing a significantly lower gene expression in HepaRG cells than in both

fresh and cryopreserved human hepatocytes. The mRNA expression data are also in

agreement with previous results on SLCO1B1 expression in HepaRG cells compared to

human hepatocytes (Kanebratt and Andersson, 2008; Le Vee et al., 2006). Furthermore, in

freshly thawed HepaRG cells in suspension, no significant OATP1B1-mediated uptake of

ATA or loss from media of E17βG was observed. The apparent inconsistent results from the

studies on ATA and E17βG regarding active uptake in HepaRG cell suspension could

possibly be explained by different experimental setup or by the variation in passive diffusion

of the two substances. When the active OATP1B1-mediated uptake of a substance is low

compared to the passive diffusion, which may be the case with the more lipophilic ATA,

HepaRG cells in suspension is not sufficiently sensitive to separate these processes

because of the low OATP1B1 expression.

In previous studies, immunohistochemical staining of the apical efflux transporters MRP2 and

MDR1 showed expression of both transporters in 3D bioreactor cultures of fresh human

hepatocytes and HepaRG cells for at least two weeks (Darnell et al., 2011; Zeilinger et al.,

2011). The localization and distribution of the transporters were similar to that found in liver

tissue. To our knowledge, our study is the first to investigate the protein expression and

activity of basolateral transporters in hepatocyte-like cells cultured in the bioreactor.

Apparently, this is also the first study to show that loss from media experiments can be

performed to evaluate the involvement of hepatic uptake transporters in the bioreactor

system using specific substrates and inhibitors. E17βG is described as a probe substrate for

both OATP1B1 and OATP1B3 (Iwai et al., 2004; Konig et al., 2000; Nakai et al., 2001;

Sasaki et al., 2004), while ATA is a clinically relevant substance that is mainly subjected to

hepatic uptake by OATP1B1 (Kameyama et al., 2005). E3S at a concentration of 30 µM is a

selective inhibitor of OATP1B1-mediated uptake with little effect on OATP1B3 (Ishiguro et al.,

2006).

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In the liver, hepatic uptake of E17βG and ATA is mediated by OATP1B1 and/or OATP1B3 as

discussed above. MRP2, and to less extent MDR1, is responsible for the biliary efflux of

E17βG, while ATA is excreted via MDR1, MRP2 and BCRP (Chen et al., 2005; Keskitalo et

al., 2009; Lau et al., 2006; Wu et al., 2000). Formation of bile structures in hepatocytes

cultured in a 3D bioreactor have been reported (Schmelzer et al., 2009; Zeilinger et al.,

2004), but the organization of these bile structures are not known and the bioreactor

technique does not allow collection of bile at this point. In the bioreactor experiments

performed in this study, intracellular E17βG is probably partly transported back into the

medium via MRP2 (and MDR1), while ATA is excreted by MDR1, MRP2 and BCRP. The

curve plateau that is observed in the loss from media experiments over time (Fig. 4 and Fig.

6) could possibly be explained by an equilibrium between uptake and efflux of E17βG and

ATA.

Lau et al. (2007) reported a 2.7-fold increase in elimination half life of ATA in vivo after oral

single dose co-administration of rifampin, a potent OATP1B1 inhibitor (and a potent CYP3A4

inducer over days). This was in line with our observations in the bioreactor experiments with

fresh human hepatocytes, where AUC of ATA increased 1.2-fold during co-administration of

E3S, another OATP1B1 inhibitor. Possible explanations for the lower interaction effect in

vitro include efflux of ATA back into the media and less potent OATP1B1 inhibition of E3S

compared to rifampin. Nevertheless, the in vitro findings demonstrate the applicability of the

bioreactor as a model for preclinical interaction studies.

Atorvastatin is a HMG-CoA reductase inhibitor administered clinically as the active acid form

(Cilla et al., 1996). In the liver, ATA and its corresponding inactive lactone form are

metabolized by CYP3A4 to 2- and 4-hydroxylated metabolites, of which the acidic forms are

also pharmacologically active (Ishigami et al., 2001; Kearney et al., 1993). In this study, ATA

was metabolized to 2-OH-ATA and 4-OH-ATA in all cell systems, demonstrating an active

CYP3A4 metabolism. These data are in agreement with studies on fresh human hepatocytes

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and HepaRG cells in bioreactor culture where CYP3A4 activity was observed for at least two

weeks (Darnell et al., 2011; Zeilinger et al., 2002), as well as studies on CYP3A4 activity in

fresh and cryopreserved human hepatocytes and HepaRG cells in suspension (Aninat et al.,

2006; Gomez-Lechon et al., 2004; Steinberg et al., 1999). Similar metabolite levels were

observed in human hepatocytes and HepaRG cells in both the bioreactor experiments at day

3 to 4 and in experiments in cell suspensions. These data are in agreement with the

comparable CYP3A4 protein expression levels observed in fresh human hepatocytes and

HepaRG cells in bioreactor culture at day 9, and with CYP3A4 mRNA expression data which

showed no significant difference in gene expression level between freshly isolated human

hepatocytes and freshly thawed cryopreserved human hepatocytes and HepaRG cells in

suspension.

In the present study, inhibition of ATA uptake by E3S in both fresh and cryopreserved human

hepatocyte suspension resulted in a significant decreased formation of ATA metabolites due

of inhibition of the OATP1B1-mediated uptake. On the contrary, in the bioreactor, an

increased metabolite formation was observed in the experiments with the inhibitor.

Atorvastastin has previously been shown to induce CYP3A4 expression in vitro (Kocarek et

al., 2002; Monostory et al., 2009). In the bioreactor, ATA actually increased CYP3A4

expression over time in the first experiment which resulted in an autoinduction in metabolism

and increased metabolite formation in the subsequent ATA plus E3S experiment in both

fresh human hepatocytes and HepaRG cells. Induction of CYP3A4 in HepaRG cells in

bioreactor culture have previously been demonstrated by Darnell et al. (2011). This study

demonstrates an induction response by ATA in both primary human hepatocytes and

HepaRG cells cultured in the bioreactor.

Variability in the quality of freshly isolated human hepatocytes is a well-known limitation of

this model. Release of ASAT and ALAT are parameters used to evaluate the quality of

hepatocytes. In this study, donor preparation 2 showed higher ASAT and ALAT values

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compared to donor preparation 1 and 3, indicating an inferior quality of the hepatocytes from

donor preparation 2. This could explain the less stable OATP1B1 activity observed in donor

preparation 2. A considerable improvement of hepatocyte cryopreservation protocols,

allowing storage, transport, scheduling of experiments and repeated experiments using

hepatocytes isolated from the same donors has been achieved during recent years. In this

study, similar data was obtained on activity and mRNA expression of both OATP1B1 and

CYP3A4 in fresh and cryopreserved human hepatocytes in suspension. These data are in

agreement with studies comparing enzyme and transporter activity in fresh and

cryopreserved human hepatocytes (Badolo et al., 2011; Hengstler et al., 2000), and shows

the improved functions and properties of cryopreserved cells (Li, 2008). In this study,

cryopreserved human hepatocytes were however not applied in the bioreactor system based

on prior experience showing that non-platable cryopreserved human hepatocytes are not

functioning in the bioreactor (data not shown). The bioreactor technique requires cell

attachment to the hollow-fiber capillary membranes, and the incapability of non-platable

cryopreserved hepatocyte to attach to surfaces probably explains the negative results. In

future experiments, platable cryopreserved human hepatocytes should be applied in the

bioreactor to test this hypothesis.

The rapid loss of enzyme and transporter activity in 2D culture of human hepatocytes is a

restriction of these systems, which prevents reliable long-term studies. For OATP1B1

substrates, uptake transport activity may have important implications for cellular drug

concentrations, which also effect drug metabolism and possible toxic effects. This study

show that freshly isolated human hepatocytes cultured in a 3D bioreactor system preserves

functionality of both OATP1B1 and CYP3A4. This allows long-term preclinical studies on

drug uptake and metabolism, which is especially important for slowly metabolized drugs.

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Acknowledgments

We thank Lisa-Mari Nilsson, Carl Jorns and Helen Zemack for technical support in the

isolation of human hepatocytes from liver tissues. Furthermore, we thank Birgitta Dillnér,

Global Safety Assessment, AstraZeneca R&D Södertälje, Sweden, for immunohistochemical

staining of OATP1B1 and CYP3A4 in bioreactor cultures of primary human hepatocytes and

HepaRG cells. We are also grateful to Dr. Bruno Stieger, Division of Clinical Pharmacology

and Toxicology, University Hospital Zurich, Switzerland, for kindly providing the human

OATP1B1 antibody.

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Authorship contributions

Participated in research design: Ulvestad, Darnell, Molden, Åsberg and Andersson

Hepatocyte isolation: Ewa Ellis

Conducted experiments: Ulvestad, Darnell

Performed data analysis: Ulvestad

Wrote or contributed to the writing of the manuscript: Ulvestad, Darnell, Molden, Åsberg and

Andersson

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Footnotes

This work was in part supported by the Research Council of Norway (ISP-FARM) [Grant

195472], the Nordic Research Board [Grant 080351], and SCR&Tox, which is funded by the

European Commission within its FP7 Programme and Cosmetics Europe, the European

Cosmetics Association, as part of the SEURAT cluster Contract number HEALTH-F5-2010-

266573.

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Figures legends

Fig. 1. (A) Bioreactor perfusion system with tubing for medium recirculation, feeding and

outflow. The recirculation pump controls the speed of medium perfusion through the

bioreactor and the feed pump supplies the system with fresh medium from the medium

bottle. The sampling port enables substance injection and sampling. (B) Multi-compartment

perfusion bioreactor with two capillary layers used for 3D culture of fresh human hepatocytes

and HepaRG cells. Each capillary layer consisted of hydrophobic oxygenation capillaries

(yellow) and hydrophilic medium perfusion capillaries (blue and red) supplying the cells

located in the inter-capillary space with oxygen/CO2 and nutrients.

Fig. 2. Time course of cell inoculation and experiments performed in fresh human

hepatocytes (n = 3) and HepaRG cells (n = 3) cultured in the bioreactor system. Loss from

media of E17βG (1 µM) +/- E3S (30 µM) was determined at day 2 and day 7 in culture, while

loss from media of ATA (5 µM) and ATA metabolite formation +/- E3S (30 µM) were

determined at day 3-4. The recirculation medium was washed out after each experiment.

Fig. 3. Time-dependent uptake of 3H-E17βG in suspension of fresh human hepatocytes (A),

cryopreserved human hepatocytes (B) and HepaRG cells (C) without (■; solid lines) and with

(□; dotted lines) co-incubation of E3S. Each point represents the mean ± S.D., n = 3. Where

the vertical error bars are not shown, the S.D. values are within the limits of the symbols.

Fig. 4. Time-dependent loss from media of 3H-E17βG in fresh human hepatocytes (A and B)

and HepaRG cells (C and D) without (■; solid lines) and with (□; dotted lines) co-incubation of

E3S at day 2 and day 7 in bioreactor culture. The medium concentration is expressed as %

of start concentration E17βG. Each point represents the mean +/- S.D., n = 2-3. Where the

vertical error bars are not shown, the S.D. values are within the limits of the symbols.

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Fig. 5. Time-dependent loss of ATA (■) and formation of ATA hydroxy metabolites (▲) in

suspension of fresh human hepatocytes (A), cryopreserved human hepatocytes (B) and

HepaRG cells (C) without (solid lines) and with (dotted lines) co-incubation of E3S. Each

point represents the mean +/- S.D., n = 3. Where the vertical error bars are not shown, the

S.D. values are within the limits of the symbols.

Fig. 6. Time-dependent loss from media of ATA in fresh human hepatocytes (A) and

HepaRG cells (B) without (■; solid lines) and with (□; dotted lines) co-incubation of E3S at

day 3-4 in bioreactor culture. The medium concentration is expressed as % of start

concentration ATA. Each point represents the mean +/- S.D., n = 3. Where the vertical error

bars are not shown, the S.D. values are within the limits of the symbols.

Fig. 7. Time-dependent ATA hydroxy metabolite formation in fresh human hepatocytes (A)

and HepaRG cells (B) without (■; solid lines) and with (□; dotted lines) co-incubation of E3S

at day 3-4 in bioreactor culture. The medium concentration is expressed as % of start

concentration ATA. Each point represents the mean +/- S.D., n = 3. Where the vertical error

bars are not shown, the S.D. values are within the limits of the symbols.

Fig. 8. Immunohistochemical staining (brown) of the basolateral uptake transporter

OATP1B1 and the metabolic enzyme CYP3A4 in bioreactor tissue of fresh human

hepatocytes from donor preparation 3 (A and C) and HepaRG cells (B and D) cultured for 9

days. OATP1B1 is evenly distributed throughout the whole cell membrane in the bioreactor

tissue. Magnification: 40-fold.

Fig. 9. mRNA expression of SLCO1B1 and CYP3A4 in fresh human hepatocytes,

cryopreserved human hepatocytes and HepaRG cells in suspension. The relative gene

expression (ΔΔCt) and the fold change of gene expression (2-∆∆Ct) were calculated using

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cryopreserved human hepatocytes as a calibrator. The bars represent the mean values ±

S.D., n = 3.

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Tables

Table 1. Human donor demographics

Donor ID Age (years) Sex (F/M) Disease Cell viability (%)

Donor 1 81 F Hepatocellular carcinoma 76

Donor 2 83 F Metastasis (colon cancer) 75

Donor 3 41 F Metastasis (rectal cancer) 70

F, female; M, male

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Table 2. AUC of medium concentration and intracellular concentration of E17βG without and with coincubation of E3S in fresh human

hepatocytes, cryopreserved human hepatocytes and HepaRG cells in suspension. The significance levels of difference in the average loss from

media or intracellular concentration of E17βG with E3S compared to without E3S are shown in the table. *p < 0.05, **p < 0.01.

Day 0 Day 0

AUC medium conc AUC intracellular conc

Cells Experiment (µmol*h/L) (pmol*h/mg protein)

Average ± S.D. Average ± S.D.

Fresh hepatocytes E17βG 893 ± 8 103 ± 7

Fresh hepatocytes E17βG + E3S (30 µM) 918 ± 3** 43 ± 13**

Cryo hepatocytes E17βG 963 ± 29 127 ± 11

Cryo hepatocytes E17βG + E3S (30 µM) 987 ± 33* 36 ± 3**

HepaRG cells E17βG 1020 ± 27 46 ± 7

HepaRG cells E17βG + E3S (30 µM) 1025 ± 40 31 ± 6**

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Table 3. AUC of medium concentration of E17βG without and with co-incubation of E3S in fresh human hepatocytes and HepaRG cells at day 2

and day 7 in bioreactor culture. The significance levels of difference in the average loss from media of E17βG with E3S compared to loss from

media of E17βG without E3S are shown in the table. *p < 0.05 and **p < 0.01.

Day 2 Day 7

Cells Experiment AUC (µmol*h/L) AUC (µmol*h/L)

BR 1 BR 2 BR 3 Average ± S.D. BR 1 BR 2 BR 3 Average ± S.D.

Fresh hepatocytes E17βG 3.49 3.25 3.37 3.37 ± 0.12 3.00 3.35 3.57 3.31 ± 0.29

Fresh hepatocytes E17βG + E3S (30 µM) 3.67 3.46 3.48 3.54 ± 0.12* 3.06 3.34 3.64 3.35 ± 0.29

HepaRG cells E17βG 4.15 4.28 4.22 ± 0.09 3.52 3.53 3.53 ± 0.01

HepaRG cells E17βG + E3S (30 µM) 4.32 4.44 4.38 ± 0.09* 3.52 3.51 3.52 ± 0.01

BR, bioreactor.

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Table 4. AUC of concentration of ATA and AUC of total concentration of 2-OH-ATA and 4-OH-ATA without and with co-incubation of E3S in

fresh human hepatocytes, cryopreserved human hepatocytes and HepaRG cells in suspension. The significance levels of difference in the

average loss of ATA and formation of ATA hydroxy metabolites with and without co-incubation of E3S are shown in the table. *p < 0.05 and **p

< 0.01.

Day 0 Day 0

Cells Experiment AUC ATA (µmol*h/L) AUC OH-ATA (µmol*h/L)

Average ± S.D. Average ± S.D.

Fresh hepatocytes ATA 152 ± 3 64 ± 20

Fresh hepatocytes ATA + E3S (30 µM) 191 ± 13* 51 ± 23*

Cryo hepatocytes ATA 164 ± 7 67 ± 17

Cryo hepatocytes ATA + E3S (30 µM) 196 ± 23* 47 ± 11*

HepaRG cells ATA 206 ± 20 52 ± 7

HepaRG cells ATA + E3S (30 µM) 207 ± 21 50 ± 6

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Table 5. AUC of medium concentration of ATA without and with co-incubation of E3S in fresh human hepatocytes and HepaRG cells at day 3-4

in bioreactor culture. The significance levels of difference in the average loss from media of E17βG with E3S compared to loss from media of

E17βG without E3S are shown in the table. *p < 0.05 and **p < 0.01. ap = 0.08.

Day 3-4

Cells Experiment AUC ATA (µmol*h/L)

Average ± S.D.

Fresh hepatocytes ATA 21.1 ± 3.2

Fresh hepatocytes ATA + E3S (30 µM) 24.6 ± 4.9a

HepaRG cells ATA 31.5 ± 5.6

HepaRG cells ATA + E3S (30 µM) 32.5 ± 7.6