药物性肝损伤(DILI)是药物开发过程中的主要问题之一。3R原理的广泛接受和体外技术的创新引入了各种新颖的模型选择,其中三维(3D)细胞球体培养在DILI预测中显示出了有希望的前景。本研究通过自组装建立了用于DILI预测的3D四细胞共培养肝球体模型。用浓度为15.42ng/mL的佛波醇12-肉豆蔻酸酯13-乙酸酯诱导48小时,随后的24小时休息期用于THP-1细胞分化,导致可靠的巨噬细胞表型。HepG2细胞,PUMC-HUVEC-T1细胞,THP-1起源的巨噬细胞,选择人肝星状细胞作为成分,在指定的球体培养条件下表现出适应性。成立后,表征证明了模型的长期稳定性的能力,反映了形态的维持,生存能力,细胞整合,和细胞-细胞连接至少六天,以及可靠的肝脏特异性功能,包括卓越的白蛋白和尿素分泌,改善药物代谢酶表达和CYP3A4活性,和MRP2,BSEP的表达,P-GP伴有胆汁酸外排转运功能。在新型3D共培养模型中使用22种DILI阳性和5种DILI阴性化合物的比较测试中,3DHepG2球体,和2DHepG2单层,与2D形式相比,3D培养方法显着增强了模型对复合细胞毒性的敏感性。新型共培养肝球体模型表现出更高的总体预测能力,具有作为分类工具的安全边际。此外,非实质细胞成分可以放大异烟肼在3D模型中的毒性,提示它们在免疫介导的毒性中的潜在介导作用。概念验证实验证明了该模型复制药物诱导的脂质失调的能力,胆汁酸外排抑制,和α-SMA上调,这是肝脏脂肪变性和磷脂变性的关键特征,胆汁淤积,和纤维化,分别。总的来说,新型3D四重细胞共培养球体模型是DILI预测的可靠且容易获得的选择。
Drug-induced liver injury (DILI) is one of the major concerns during drug development. Wide acceptance of the 3 R principles and the innovation of in-vitro techniques have introduced various novel model options, among which the three-dimensional (3D) cell spheroid cultures have shown a promising prospect in DILI prediction. The present study developed a 3D quadruple cell co-culture liver spheroid model for DILI prediction via self-assembly. Induction by phorbol 12-myristate 13-acetate at the concentration of 15.42 ng/mL for 48 hours with a following 24-hour rest period was used for THP-1 cell differentiation, resulting in credible macrophagic phenotypes. HepG2 cells, PUMC-HUVEC-T1 cells, THP-1-originated macrophages, and human hepatic stellate cells were selected as the components, which exhibited adaptability in the designated spheroid culture conditions. Following establishment, the characterization demonstrated the competence of the model in long-term stability reflected by the maintenance of morphology, viability, cellular integration, and cell-cell junctions for at least six days, as well as the reliable liver-specific functions including superior albumin and urea secretion, improved drug metabolic enzyme expression and CYP3A4 activity, and the expression of MRP2, BSEP, and P-GP accompanied by the bile acid efflux transport function. In the comparative testing using 22 DILI-positive and 5 DILI-negative compounds among the novel 3D co-culture model, 3D HepG2 spheroids, and 2D HepG2 monolayers, the 3D culture method significantly enhanced the model sensitivity to compound cytotoxicity compared to the 2D form. The novel co-culture liver spheroid model exhibited higher overall predictive power with margin of safety as the classifying tool. In addition, the non-parenchymal cell components could amplify the toxicity of isoniazid in the 3D model, suggesting their potential mediating role in immune-mediated toxicity. The proof-of-concept experiments demonstrated the capability of the model in replicating drug-induced lipid dysregulation, bile acid efflux inhibition, and α-SMA upregulation, which are the key features of liver steatosis and phospholipidosis, cholestasis, and fibrosis, respectively. Overall, the novel 3D quadruple cell co-culture spheroid model is a reliable and readily available option for DILI prediction.