Zimmermann Andrea, Scheffschick Andrea, Hänsel René, Borchardt Hannes, Liu Jia Li, Ehnert Sabrina, Schicht Gerda, Seidemann Lena, Aigner Achim, Schiffmann Susanne, Nüssler Andreas, Seehofer Daniel, Damm Georg
Department of Hepatobiliary Surgery and Visceral Transplantation, Clinic and Polyclinic for Visceral, Transplant, Thoracic and Vascular Surgery, Leipzig University Medical Center, Leipzig, Germany.
Saxonian Incubator for Clinical Translation (SIKT), University of Leipzig, Leipzig, Germany.
Arch Toxicol. 2025 Mar;99(3):1167-1185. doi: 10.1007/s00204-024-03943-8. Epub 2024 Dec 22.
The development of in vitro hepatocyte cell culture systems is crucial for investigating drug-induced liver injury (DILI). One prerequisite for monitoring DILI related immunologic reactions is the extension of primary human hepatocyte (PHH) cultures towards the inclusion of macrophages. Therefore, we developed and characterized an autologous co-culture system of PHH and primary human hepatic macrophages (hepM) (CoC1). We compared CoC1 with a co-culture of the same PHH batch + M0 macrophages derived from THP1 cells (CoC2) in order to represent a donor independent macrophage reaction. Then, we treated the mono- and co-cultures with drugs that cause DILI-menadione (MEN, 1 or 10 µM, 3 h), diclofenac (DIC, 0.5 or 5 mM, 6 h), or acetaminophen (APAP, 0.5 or 5 mM, 6 h)-and assessed culture stability, cell activity, macrophage differentiation, cytokine production and cell viability. Without drug treatment, CoC1 was the most stable over a culture time of up to 60 h. Cytokine array analysis revealed a proinflammatory profile of PHH mono-cultures due to isolation stress but showed different influences of hepM and M0 on the cytokine profile in the co-cultures. MEN, DIC and APAP treatment led to donor-dependent signs of cell stress and toxicity. HepM can either promote or reduce the DILI effects donor dependently in CoC1. CoC2 are slightly less sensitive than CoC1 in representing DILI. In summary, we present a new autologous co-culture system that can mimic DILI in a donor-dependent manner. This cellular system could be useful for new drug testing strategies and reducing animal testing.
体外肝细胞培养系统的发展对于研究药物性肝损伤（DILI）至关重要。监测与DILI相关免疫反应的一个前提条件是将原代人肝细胞（PHH）培养扩展至纳入巨噬细胞。因此，我们开发并表征了一种PHH与原代人肝巨噬细胞（hepM）的自体共培养系统（CoC1）。我们将CoC1与同一批PHH + 源自THP1细胞的M0巨噬细胞的共培养物（CoC2）进行比较，以代表不依赖供体的巨噬细胞反应。然后，我们用导致DILI的药物（维生素K3（MEN，1或10 μM，3小时）、双氯芬酸（DIC，0.5或5 mM，6小时）或对乙酰氨基酚（APAP，0.5或5 mM，6小时））处理单培养物和共培养物，并评估培养稳定性、细胞活性、巨噬细胞分化、细胞因子产生和细胞活力。在无药物处理的情况下，CoC1在长达60小时的培养时间内是最稳定的。细胞因子阵列分析显示，由于分离应激，PHH单培养物具有促炎特征，但显示hepM和M0对共培养物中细胞因子谱有不同影响。MEN、DIC和APAP处理导致了依赖供体的细胞应激和毒性迹象。在CoC1中hepM可依供体不同促进或降低DILI效应。CoC2在代表DILI方面比CoC1稍不敏感。总之，我们提出了一种新的自体共培养系统，其可以以依赖供体的方式模拟DILI。这种细胞系统可能对新的药物测试策略和减少动物实验有用。
