In addition to hepatocytes, the liver comprises a host of specialised non-parenchymal cells which are important to consider in the development of in vitro models which are both physiologically and toxicologically relevant. We have characterized a 3D co-culture system comprising primary human hepatocytes (PHH) and non-parenchymal cells (NPC) and applied it to the investigation of acetaminophen-induced toxicity. Firstly, we titrated ratios of PHH:NPC and confirmed the presence of functional NPCs via both immunohistochemistry and activation with both LPS and TGF-β. Based on these data we selected a ratio of 2:1 PHH:NPC for further studies. We observed that spheroids supplemented with NPCs were protected against acetaminophen (APAP) toxicity as determined by ATP (up to threefold difference in EC50 at day 14 compared to hepatocytes alone) and glutathione depletion, as well as miR-122 release. APAP metabolism was also altered in the presence of NPCs, with significantly lower levels of APAP-GSH detected. Expression of several CYP450 enzymes involved in the bioactivation of APAP was also lower in NPC-containing spheroids. Spheroids containing NPCs also expressed higher levels of miRNAs which have been implicated in APAP-induced hepatotoxicity, including miR-382 and miR-155 which have potential roles in liver regeneration and inflammation, respectively. These data indicate that the interaction between hepatocytes and NPCs can have significant metabolic and toxicological consequences important for the correct elucidation of hepatic safety mechanisms.

中文翻译：

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3D 球体模型中原发性肝非实质细胞的功能和对乙酰氨基酚肝毒性的贡献。

除肝细胞外，肝脏还包含大量专门的非实质细胞，这些细胞在开发具有生理学和毒理学相关性的体外模型时非常重要。我们已经描述了一个包含原代人肝细胞 (PHH) 和非实质细胞 (NPC) 的 3D 共培养系统，并将其应用于对乙酰氨基酚诱导的毒性研究。首先，我们滴定了 PHH:NPC 的比例，并通过免疫组织化学和 LPS 和 TGF-β 的激活证实了功能性 NPC 的存在。基于这些数据，我们选择了 2:1 PHH:NPC 的比例进行进一步研究。我们观察到，通过 ATP（与单独的肝细胞相比，第 14 天的 EC50 差异高达三倍）和谷胱甘肽消耗以及 miR-122 释放，补充了 NPC 的球状体免受对乙酰氨基酚 (APAP) 毒性的影响。在 NPC 存在的情况下，APAP 代谢也发生了变化，检测到的 APAP-GSH 水平显着降低。在含有 NPC 的球体中，参与 APAP 生物活化的几种 CYP450 酶的表达也较低。含有 NPC 的球体也表达更高水平的 miRNA，这些 miRNA 与 APAP 诱导的肝毒性有关，包括分别在肝再生和炎症中具有潜在作用的 miR-382 和 miR-155。