Primary mouse hepatocytes isolated from genetically defined and/or diverse lines and disease models are a valuable resource for studying the impact of genetic and environmental factors on drug response and disease. However, standard monolayer cultures result in a rapid decline in mouse hepatocyte viability and functionality. Therefore, we evaluated 3D spheroid methodology for long-term culture of primary mouse hepatocytes, initially to support investigations of drug-induced liver injury (DILI). Primary hepatocytes isolated from male and female C57BL/6J mice were used to generate spheroids by spontaneous self-aggregation in ultra-low attachment plates. Spheroids with well-defined perimeters were observed within 5 days after seeding and retained morphology, ATP, and albumin levels for an additional 2 weeks in culture. Global microarray profiling and quantitative targeted proteomics assessing 10 important drug metabolizing enzymes and transporters demonstrated maintenance of mRNA and protein levels in spheroids over time. Activities for 5 major P450 enzymes were also stable and comparable to activities previously reported for human hepatocyte spheroids. Time- and concentration-dependent decreases in ATP and albumin were observed in response to the DILI-causing drugs acetaminophen, fialuridine, AMG-009, and tolvaptan. Collectively, our results demonstrate successful long-term culture of mouse hepatocytes as spheroids and their utility to support investigations of DILI.

从遗传定义和/或不同的系和疾病模型中分离的原代小鼠肝细胞是研究遗传和环境因素对药物反应和疾病的影响的宝贵资源。然而，标准单层培养会导致小鼠肝细胞活力和功能迅速下降。因此，我们评估了用于原代小鼠肝细胞长期培养的 3D 球体方法，最初是为了支持对药物性肝损伤 (DILI) 的研究。从雄性和雌性 C57BL/6J 小鼠中分离出的原代肝细胞用于通过超低附着板中的自发自聚集来生成球体。在播种后 5 天内观察到具有明确周长的球体，并在培养中再保留 2 周的形态、ATP 和白蛋白水平。全球微阵列分析和定量靶向蛋白质组学评估了 10 种重要的药物代谢酶和转运蛋白，表明随着时间的推移，球体中的 mRNA 和蛋白质水平保持不变。5 种主要 P450 酶的活性也很稳定，与之前报道的人类肝细胞球体的活性相当。对引起 DILI 的药物对乙酰氨基酚、氟尿苷、AMG-009 和托伐普坦，观察到 ATP 和白蛋白的时间和浓度依赖性降低。总的来说，我们的结果证明了小鼠肝细胞作为球体的成功长期培养及其在支持 DILI 研究中的效用。5 种主要 P450 酶的活性也很稳定，与之前报道的人类肝细胞球体的活性相当。对引起 DILI 的药物对乙酰氨基酚、氟尿苷、AMG-009 和托伐普坦，观察到 ATP 和白蛋白的时间和浓度依赖性降低。总的来说，我们的结果证明了小鼠肝细胞作为球体的成功长期培养及其在支持 DILI 研究中的效用。5 种主要 P450 酶的活性也很稳定，与之前报道的人类肝细胞球体的活性相当。对引起 DILI 的药物对乙酰氨基酚、氟尿苷、AMG-009 和托伐普坦，观察到 ATP 和白蛋白的时间和浓度依赖性降低。总的来说，我们的结果证明了小鼠肝细胞作为球体的成功长期培养及其在支持 DILI 研究中的效用。