Oorts Marlies, Richert Lysiane, Annaert Pieter
Drug Delivery and Disposition, KU Leuven, Department of Pharmaceutical and Pharmacological Sciences, O&N2, Herestraat 49, Box 921, 3000 Leuven, Belgium.
KaLy-Cell, 20A rue du Général Leclerc, 67115 Plobsheim, France; Université de Franche-Comté, 25030 Besançon, France.
J Pharmacol Toxicol Methods. 2015 May-Jun;73:63-71. doi: 10.1016/j.vascn.2015.03.002. Epub 2015 Mar 28.
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.
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).
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.
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.
在药物研发中，体外鉴定可在体内引起胆汁淤积的化合物仍是一个问题。现有的体外系统对临床情况的预测性较差。最近，我们的研究小组开发了一种基于三明治培养（大鼠）肝细胞（SC(R)H）的模型，用于通过改变胆汁酸（BA）稳态来检测引起胆汁淤积的化合物（Chatterjee等人，2014年）。在本研究中，我们评估了该模型在新鲜分离和冷冻保存的肝细胞中表现是否同样良好。
我们将冷冻保存前后的大鼠肝细胞三明治培养物暴露于胆汁淤积化合物环孢素A（CsA）和曲格列酮（Tro），分别在有和没有BA混合物的情况下。孵育结束时，测量肝细胞产生尿素的能力，以确定药物诱导的胆汁淤积指数（DICI）的变化。
与冷冻保存肝细胞的培养物（22.86（±1.91）nmol尿素/cm²）相比，新鲜分离肝细胞的三明治培养物中平均（±SEM）尿素产量显著更高（27.88（±0.96）nmol/cm²）。然而，在根据汇合率（基于光学显微镜图像分析）进行标准化后，似乎所有批次的SCRH的尿素产量相似。10μM CsA和75μM Tro的平均（±SEM）DICI值分别为0.89（±0.03）和0.93（±0.03）。与单独使用化合物相比，更高浓度的CsA（≥15μM）和Tro（≥100μM）在与BA混合物一起孵育时会导致尿素产量显著降低。所有批次的SCRH都是如此，无论冷冻保存历史如何。
总之，当将先前描述的体外胆汁淤积模型应用于冷冻保存前后的SCRH时，未观察到显著差异。本研究证明了该模型的稳健性，这意味着它可用于从新鲜分离和冷冻保存的肝细胞获得的SCRH。
