Drug-induced cholestasis detection in cryopreserved rat hepatocytes in sandwich culture

Introduction: In vitro identification of compounds that cause cholestasis in vivo still remains a problemin pharmaceutical R&D. Currently existing in vitro systems show poor predictivity towards the clinical situation. Recently, our research group developed a model, based on sandwich-cultured (rat) hepatocytes (SC(R) H), to detect compounds causing cholestasis via altered bile acid (BA) homeostasis (Chatterjee et al., 2014). In the present study, we assessed whether this model performs equally well with freshly-isolated and cryopreserved hepatocytes. Methods: We exposed sandwich cultures from rat hepatocytes before and after cryopreservation to the cholestatic compounds, cyclosporin A (CsA) and troglitazone (Tro), in the presence and in the absence of a BA mixture. At the end of the incubations, the capability of the hepatocytes to produce urea was measured to determine changes in the drug-induced cholestasis index (DICI). Results: The mean (+/- SEM) urea production was significantly higher in sandwich cultures from freshlyisolated hepatocytes (27.88 (+/- 0.96) nmol/cm(2)), compared to cultures from cryopreserved hepatocytes (22.86 (+/- 1.91) nmol urea/cm(2)). However, after normalization for confluence rate (based on light microscopic image analysis), it appeared that the urea production was similar for all the batches of SCRH. The mean (+/- SEM) DICI values for CsA 10 mu Mand Tro 75 mu Mwere 0.89 (+/- 0.03) and 0.93 (+/- 0.03), respectively. Higher concentrations, CsA (>= 15 mu M) and Tro (>= 100 mu M), elicited a significant decrease in urea production when incubated in the presence of a BA mixture compared to the compound alone. This was the case for all the batches of SCRH, irrespective of cryopreservation history. Discussion: In conclusion, no significant differenceswere seenwhen the previously described in vitro cholestasis model was applied in SCRH before or after cryopreservation. This study demonstrates the robustness of the model, which implies that it can be used with SCRH obtained from both freshly-isolated and cryopreserved hepatocytes. (C) 2015 Elsevier Inc. All rights reserved.