title: Drug-induced cholestasis detection in cryopreserved rat hepatocytes in sandwich culture.
abstract: BACKGROUND
  In vitro identification of compounds that cause cholestasis in vivo still remains a problem in 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 freshly-isolated 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 μM and Tro 75 μM were 0.89 (± 0.03) and 0.93 (± 0.03), respectively. Higher concentrations, CsA (≥ 15 μM) and Tro (≥ 100 μM), 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.
  
  
  CONCLUSIONS
  In conclusion, no significant differences were seen when 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.
authors.affiliation: Drug Delivery and Disposition, KU Leuven, Department of Pharmaceutical and Pharmacological Sciences, O&N2, Herestraat 49, Box 921, 3000 Leuven, Belgium. Electronic address: Marlies.Oorts@pharm.kuleuven.be.
authors.forename: Marlies
authors.initials: M
authors.lastname: Oorts
authors.affiliation: KaLy-Cell, 20A rue du Général Leclerc, 67115 Plobsheim, France; Université de Franche-Comté, 25030 Besançon, France. Electronic address: l.richert@kaly-cell.com.
authors.forename: Lysiane
authors.initials: L
authors.lastname: Richert
authors.affiliation: Drug Delivery and Disposition, KU Leuven, Department of Pharmaceutical and Pharmacological Sciences, O&N2, Herestraat 49, Box 921, 3000 Leuven, Belgium. Electronic address: Pieter.Annaert@pharm.kuleuven.be.
authors.forename: Pieter
authors.initials: P
authors.lastname: Annaert
mesh_terms: D000818:Animals; D001647:Bile Acids and Salts / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002779:Cholestasis / Q000139:chemically induced* / Q000473:pathology; D002839:Chromans / Q000633:toxicity*; D015925:Cryopreservation / Q000379:methods; D016572:Cyclosporine / Q000633:toxicity*; D022781:Hepatocytes / Q000187:drug effects* / Q000473:pathology; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D045162:Thiazolidinediones / Q000633:toxicity*; D000077288:Troglitazone
chemical_list: D001647:Bile Acids and Salts; D002839:Chromans; D045162:Thiazolidinediones; D016572:Cyclosporine; D000077288:Troglitazone
keywords: Cryopreserved hepatocytes; Cyclosporin A; Drug-induced cholestasis; Sandwich-cultured rat hepatocytes; Troglitazone