Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China; National Center for Safety Evaluation of Drugs, National Institutes for Food and Drug Control, Beijing Key Laboratory for Nonclinical Safety Evaluation of Drugs, Beijing 100176, China.
National Center for Safety Evaluation of Drugs, National Institutes for Food and Drug Control, Beijing Key Laboratory for Nonclinical Safety Evaluation of Drugs, Beijing 100176, China.
Toxicology. 2024 Jun;505:153829. doi: 10.1016/j.tox.2024.153829. Epub 2024 May 11.
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.
药物性肝损伤（DILI）是药物开发过程中的主要关注点之一。广泛接受 3R 原则和创新的体外技术已经引入了各种新的模型选择，其中三维（3D）细胞球体培养在 DILI 预测中显示出有前途的前景。本研究通过自组装开发了一种用于 DILI 预测的 3D 四重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 13-乙酸诱导 48 小时，随后休息 24 小时，用于 THP-1 细胞分化，产生可信的巨噬细胞表型。HepG2 细胞、PUMC-HUVEC-T1 细胞、THP-1 衍生的巨噬细胞和人肝星状细胞被选为成分，它们在指定的球体培养条件下表现出适应性。建立后，该模型的特征表现为至少六天的长期稳定性，包括形态、活力、细胞整合和细胞-细胞连接的维持，以及可靠的肝脏特异性功能，包括优异的白蛋白和尿素分泌、改善药物代谢酶表达和 CYP3A4 活性以及 MRP2、BSEP 和 P-GP 的表达，并伴有胆汁酸外排转运功能。在使用新型 3D 共培养模型中的 22 种 DILI 阳性和 5 种 DILI 阴性化合物进行比较测试时，与 2D 形式相比，3D 培养方法显著提高了模型对化合物细胞毒性的敏感性。新型共培养肝球体模型以安全性作为分类工具，表现出更高的整体预测能力。此外，非实质细胞成分可以在 3D 模型中放大异烟肼的毒性，表明它们在免疫介导的毒性中可能具有潜在的介导作用。概念验证实验表明，该模型能够复制药物诱导的脂质失调、胆汁酸外排抑制和α-SMA 上调，这分别是肝脂肪变性和磷脂沉积、胆汁淤积和纤维化的关键特征。总的来说，新型 3D 四重细胞共培养球体模型是一种可靠且易于获得的 DILI 预测选择。
