POSTERS PHHs in vitro restrict their use; whilst cell lines such as HepG2/C3As lack a substantial and variable set of liver-specific functions, specifically, CYP450 activity. In this study we compared CYP450 activity/metabolism between HepG2/C3A and human HepaRG cells as hepatic models for pre-clinical drug testing. Methods: Human hepatic cell lines [HepG2/C3A or HepaRG] were grown to >80% confluence on collagen-I-coated plates and treated (in triplicates) 24 h with prototypical inducers rifampicin (CYP3A4) and omeprazole (CYP1A2), [n = 3]. 50mM testosterone or phenacetin were added and supernatant and cell samples taken after 2 hours of incubation at 37°C. CYP1A2/3A4 activity [P450-Glo™-Luminometry; Promega] was determined (Figure 1). Relative turnover of testosterone [HPLC] and phenacetin [LC- MS/MS] metabolites was also measured. Cell phenotype was assessed by light-microscopy, histology (PAS-Glycogen), CYP3A4, F-actin/phalloidin, and JC-1 fluorescent-staining. Results: Figure 1 shows HepaRG CYP1A2/3A4 activity was 40–80 fold >> HepG2/C3A cells [P < 0.001]; Drug profiling revealed HepaRGs had both enhanced production of major metabolites of phenacetin and testosterone and more intact drug metabolism compared with HepG2/C3A. In contrast with HepG2/C3A, HepaRGs displayed a more intact hepatic phenotype, including: Strong positive glycogen, CYP3A4 staining, high JC-1-positive intrinsic metabolic activity (DYm) and organotypic gross morphology. Figure 1. Relative activity of CYP1A2 and CYP3A4 enzymes in HepaRG and C3A cells under induction by omeprazole (OMP, CY1A2) or rifampicin (RIF, CYP3A4) controlled against additional enzyme-specific inhibition by fluvoxamin (FLU, CYP1A2) or ketoconazole (KETO, CYP3A4). Activity was measured by luminometry and was normalized internally against appropriate controls. Data are displayed as mean ± standard deviation of % relative activity, normalized to the activity of HepaRG cells under induction (= 100%) and derived from n = 3 biological and n = 8 technical replicates each. Conclusions: HepaRG cells may represent a more physiologically- relevant pre-clinical platform for CYP450 activation/inhibition, safety pharmacology, as well as drug-drug interaction studies. P332 DEVELOPMENT OF HUMAN HEPATIC HepaRG CELL-BASED LiverBioChip PROTOTYPES USING ELECTRON BEAM LITHOGRAPHY DIRECTED-NANOPATTERNING FOR HTS- COMPATIBLE PRE-CLINICAL DRUG EVALUATION APPLICATIONS L.J. Nelson 1 , C. LeBled 1 , P. Treskes 1 , M. Thompson 2 , M. Navarro 1 , J.N. Plevris 1 , N. Gadegaard 2 . 1 Hepatology, University of Edinburgh, Edinburgh, 2 James Watt Nanofabrication Centre, University of Glasgow, Glasgow, United Kingdom E-mail: lnelson1@staffmail.ed.ac.uk Background and Aims: Cells are known to respond to nanoscale features thus our aim was to assess phenotypic morphology of HepaRGs cultured on 2D-nanopatterned polymer slides (NPS) for the optimisation of prototype LiverBioChips for pre-clinical drug- screening. Methods: NPS were manufactured using electron-beam lithography and injection moulding. Slides comprised 11 discrete areas representing varying degrees of nanoscale symmetry and disorder (including 1x planar surface as control). Human hepatic HepaRG and HepG2/C3A control cells were seeded in culture media, on NPS substrates at high (standard; 2.4×10 5 /cm 2 or 5.6×10 4 /cm 2 respectively) intermediate (75% of high) and low (50%) seeding densities. Cells were cultured for up to 6 days; and morphological phenotype analyzed on days 3 and 6, using light microscopy; LIVE/DEAD viability-assay and ‘Profiler Panel’ immunostaining comprising CYP3A4/phalloidin- TRITC/DAPI fluorochromes. Captured images were analysed for cell morphometrics using CellProfiler software. Results: HepaRG cells on two different nanotopographies displayed clear differences in cell attachment/-spreading and enhanced maturation (Figure 1). We observed pattern-dependent early appearance of bile-canaliculae. Moreover, HepaRG differentiated phenotype was observed earlier (day 3) and at lower (intermediate) seeding densities compared with high-density HepaRGs cultured on standard collagen-I substrate (day 6). In contrast HepG2/C3As at all seeding densities did not respond to nanopatterning, maintaining a typical phenotype. Preliminary image analysis of cell morphometrics, could delineate cell numbers/neighbours, as well as relative area/intensity of CYP3A4 staining in HepaRG cultures. Figure 1. HepaRG cells seeded on two different nanotopographies (DSQ or HEX patterns as indicated) displaying clear differences in proliferation rate and enhanced lineage determination suggesting relative differentiation/ cells numbers are influenced by nanopatterned substrates. Shown are side-by-side displays of HepaRG cells after indicated nanopattern-culture and dual staining via DAPI and phalloidin at 20× magnification and after triple staining including CYP3A4 at 10× magnification. Note punctuate phalloidin-staining of F-actin bands indicative of bile-canaliculae. Conclusions: HepaRG-based 2D directed-nanopatterning coupled with CellProfiler analysis represents proof-of-concept for developing prototype LiverBiochips, as a convenient HTS- compatible method for assessing multiparametric cell descriptors for drug development. P333 HUMAN HEPATIC HepaRG CO-CULTURE MODEL AS A SENSITIVE AND NON-INVASIVE TOXICOLOGICAL PLATFORM USING ECIS (ELECTRICAL CELL-SUBSTRATE IMPEDANCE SENSING) BIOSENSORS W. Gamal 1 , P. Treskes 2 , C. Chesne 3 , J.N. Plevris 2 , P.-O. Bagnaninchi 1 , L.J. Nelson 2 . 1 Centre for Biomedical Engineering, 2 Hepatology, University of Edinburgh, Edinburgh, United Kingdom; 3 Biopredic International, Rennes, France E-mail: lnelson1@staffmail.ed.ac.uk Background and Aims: A robust HTS-compatible multiparametric platform for assessing drug hepatotoxicity would provide a new paradigm for in vitro toxicology. Using the highly-differentiated human hepatic cell line HepaRG as a surrogate to primary human hepatocytes, our aim was to profile real-time changes in cellular impedance, Z , in response to paracetamol toxicity. Such dynamic screening allows capture and modeling of multiparametric, high- content data using ECIS biosensors. Methods: HepaRG cells were cultured to confluence (terminally- differentiated Hepatocyte:Cholangiocyte co-culture) on ECIS- 8W10+ and 8W1E-dd 8-well (250,000 cells/well) electrode arrays. Following CYP3A4 induction (day 8), with Rifampicin (24h), a paracetamol time-/dose-response [DMSO-vehicle as controls] was monitored with Impedance Z measurements taken every 180 sec Journal of Hepatology 2014 vol. 60 | S67–S214 S177