Development of 2D organotypic rat liver co-culture models for assessing hepatotoxicant modes of action

In both the regulatory and commercial arenas, the goal is to move away from time consuming, costly in-life rodent studies and towards safety assessment strategies that rely on testing species-relevant cells in vitro. The liver has been a major focus of these efforts, yet there are currently no in vitro alternatives for hepatotoxicity testing accepted by regulators, and the assays that do exist typically utilize hepatocyte monolayer culture. While hepatocytes have been the primary component of in vitro hepatotoxicity assay development, the non-parenchymal cells (NPCs) (i.e., hepatic stellate cells, Kupffer cells, and liver sinusoidal endothelial cells) also play a critical role in the progression of liver pathologies. The goal of this study was to develop an organotypic co-culture system that could read out the various mechanisms of action observed in vivo, as well as maintain metabolic capability and extended viability to allow for repeat dosing. We developed a two-dimensional 96-well plate-based co-culture system that includes primary rat hepatocytes, stellate, Kupffer, and endothelial cells that supports hepatocyte viability and phenotype stability for up to eight days. Importantly, markers of hepatocyte differentiation and polarity are maintained in co-culture, but hepatocyte monocultures with no other cell types lose phenotypic markers after prolonged culture. This co-culture model was leveraged to assess the effects of known hepatotoxic stimuli, and the co-culture model resulted in responses that were more representative of the in vivo phenotypes.在监管与商业双领域中，当前的核心目标均已从耗时耗财的活体啮齿类动物实验，转向依托体外（in vitro）检测物种相关细胞的安全性评价策略。肝脏一直是这类研究的重点方向，但目前尚无被监管机构认可的体外肝毒性（hepatotoxicity）检测替代方案，现有检测方法多采用肝细胞（hepatocyte）单层培养。尽管肝细胞始终是体外肝毒性检测方法开发的核心组分，非实质细胞（non-parenchymal cells, NPCs）——即肝星状细胞（hepatic stellate cells）、库普弗细胞（Kupffer cells）与肝窦内皮细胞（liver sinusoidal endothelial cells）——在肝脏病理进程中也发挥着关键作用。本研究旨在开发一种器官型共培养体系（organotypic co-culture system），该体系可复现体内（in vivo）观察到的多种作用机制，同时维持代谢能力与较长的细胞存活时长，以支持重复给药实验。我们构建了基于二维96孔板（96-well plate）的共培养体系，包含原代大鼠肝细胞（primary rat hepatocytes）、肝星状细胞、库普弗细胞与内皮细胞，可维持肝细胞存活与表型稳定长达8天。值得注意的是，该共培养体系可保留肝细胞分化与极性标志物，而不含其他细胞类型的肝细胞单细胞培养在长期培养后会丢失相关表型标志物。我们利用该共培养模型评估了已知肝毒性刺激物的作用效果，结果显示该模型的应答更贴合体内表型。