Comparative Localization and Functional Activity of the Main Hepatobiliary Transporters in HepaRG Cells and Primary Human Hepatocytes

Abstract

The role of hepatobiliary transporters in drug-induced liver injury remains poorly understood. Various in vivo and in vitro biological approaches are currently used for studying hepatic transporters; however, appropriate localization and functional activity of these transporters are essential for normal biliary flow and drug transport. Human hepatocytes (HHs) are considered as the most suitable in vitro cell model but erratic availability and inter-donor functional variations limit their use. In this work, we aimed to compare localization of influx and efflux transporters and their functional activity in differentiated human HepaRG hepatocytes with fresh HHs in conventional (CCHH) and sandwich (SCHH) cultures. All tested influx and efflux transporters were correctly localized to canalicular [bile salt export pump (BSEP), multidrug resistance-associated protein 2 (MRP2), multidrug resistance protein 1 (MDR1), and MDR3] or basolateral [Na(+)-taurocholate co-transporting polypeptide (NTCP) and MRP3] membrane domains and were functional in all models. Contrary to other transporters, NTCP and BSEP were less abundant and active in HepaRG cells, cellular uptake of taurocholate was 2.2- and 1.4-fold and bile excretion index 2.8- and 2.6-fold lower, than in SCHHs and CCHHs, respectively. However, when taurocholate canalicular efflux was evaluated in standard and divalent cation-free conditions in buffers or cell lysates, the difference between the three models did not exceed 9.3%. Interestingly, cell imaging showed higher bile canaliculi contraction/relaxation activity in HepaRG hepatocytes and larger bile canaliculi networks in SCHHs. Altogether, our results bring new insights in mechanisms involved in bile acids accumulation and excretion in HHs and suggest that HepaRG cells represent a suitable model for studying hepatobiliary transporters and drug-induced cholestasis.

Alteration of human hepatic drug transporter activity and expression by cigarette smoke condensate

“…Human highly-differentiated hepatoma HepaRG cells, which express most of hepatic drug transporters (Kotani et al 2012;Le Vee et al 2013) and are therefore well-recognized as surrogates of human hepatocytes in drug transporter studies (Bachour-El Azzi et al 2015), were routinely cultured in Williams' E medium (Life Technologies) supplemented with 10% (vol/vol) fetal calf serum, 10 IU/mL penicillin, 10 μg/mL streptomycin, 5 μg/mL insulin, 2 mM glutamine, and 5 x 10 −5 M hydrocortisone hemisuccinate. Additional culture for two weeks in the same medium supplemented with 2% (vol/vol) DMSO was performed in order to get a full hepatocytic differentiation of the cells (Gripon et al 2002).…”

A dynamic mathematical model of bile acid clearance in HepaRG cells

“…Time-dependent increased accumulation in the medium during the 30min incubation likely reflected an increased sinusoidal efflux associated with the activation of the MRP3 membrane transporter in the presence of drugs, which has been described as a compensatory mechanism in the event of BSEP inhibition (Yang et al, 2013). In support of this, immuno-localization of MRP3 showed a more intense labeling of the sinusoidal membrane after 30min of CsA treatment (Sharanek, et al, 2015). However, it cannot be excluded that a fraction of TCA was effluxed to the supernatant via BC.…”

“…Moreover, these biological donor-donor variabilities associated with PHH may confound opportunities to sequentially build an integrated time-resolved mathematical model using a singularly phenotypically stable, reproducible and robust in vitro model by which to describe bile acid clearance and flow, and study the impact of drug effects with time 4 in culture. By contrast, the HepaRG cell line exhibits a well-characterized and stable hepatocyte-like phenotype (Guguen-Guillouzo and Guillouzo, 2010) by which to reproducibly measure bile formation and bile canalicular (BC) clearance (Sharanek et al, 2015), to establish a dynamic mathematical model describing bile acid clearance.…”

Inhibition of bile canalicular network formation in rat sandwich cultured hepatocytes by drugs associated with risk of severe liver injury

“…To overcome these limitations, other cell sources, such as cryopreserved human hepatocytes, HepaRG, and HepG2 cells, will be considered in the future because all these cells exhibit polarized phenotype with distinct BC structures (Zegers and Hoekstra, 1998;Guillouzo et al, 2007). Among these three options, human hepatocytes can exhibit time-dependent extension of BC networks (Hoffmaster et al, 2004), while HepaRG and HepG2, which are both immortalized human hepatocytes, have not yet been examined or are unable to form extended BC network in standard sandwich culture conditions (Bachour-El Azzi et al, 2015;Herrema et al, 2006). Culture devices other than a sandwich configuration such as spheroid culture, microfluidic systems (Ebrahimkhani et al, 2014), or application of human induced pluripotent stem cell-derived hepatocytes (Godoy et al, 2013) should be considered.…”