Holm Camden, Finelli Joseph, Frare Mateo, Armento Alex, Ayehunie Seyoum
MatTek Corporation, Ashland, MA, United States.
Front Toxicol. 2025 Jun 12;7:1574387. doi: 10.3389/ftox.2025.1574387. eCollection 2025.
In drug development, liver failure is the cause of approximately 30% of post marketing withdrawals of pharmaceuticals. Drug-induced liver injury (DILI) remains the leading cause of acute liver failure (ALF), accounting for approximately 15% of the cases.
In this study, we developed a novel human three-dimensional (3D) liver tissue model by seeding adult primary human hepatocytes onto cell culture inserts under Air-Liquid Interface (ALI) condition for extended culture periods. The engineered tissues were thoroughly characterized for barrier integrity using transepithelial electrical resistance (TEER) measurements and assessed for tissue morphology and structure via hematoxylin and eosin (H&E) staining and immunohistochemistry. Expression levels of drug transporters and drug-metabolizing enzymes were evaluated by quantitative PCR (qPCR). The functionality of the tissue model for drug toxicity assessment was demonstrated by comparison with conventional two-dimensional (2D) monolayer hepatocyte cultures and liver spheroids. To evaluate the model's relevance for DILI studies, we exposed the 3D liver tissues to compounds with well-documented hepatotoxic profiles in humans. Liver function was monitored by quantifying biomarkers such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) released into the culture medium.
The engineered 3D liver tissue model exhibited distinct apical and basolateral surfaces, reflecting a polarized and stratified architecture that closely mimics native liver tissue. Morphological and phenotypic analyses confirmed the tissue's organotypic features. Gene expression profiling revealed elevated levels of liver-specific genes involved in drug transport, metabolism, and clearance. Functionally, the tissue metabolized midazolam--a substrate of the cytochrome P450 3A4 (CYP3A4) enzyme--into its primary metabolite, 1-hydroxymidazolam. Upon repeated exposure to fialuridine, a discontinued anti-hepatitis B drug known for causing severe liver toxicity in humans, the tissue model exhibited barrier compromise, reduced albumin production, and increased levels of ALT and AST in a time- and concentration-dependent manner.
The results strongly suggest the model's physiological relevance and functionality in predicting drug responses in humans. Thus, the engineered 3D organotypic human liver tissue model which can be cultured for weeks and produced in a semi-high throughput format creates an opportunity to study drug-induced liver toxicity in an in vitro microenvironment. The reconstructed 3D liver tissue model can serve as a tool for alternative methods intended to reduce animal use in experimentation.
在药物研发过程中，肝衰竭是约30%的药品上市后撤市的原因。药物性肝损伤（DILI）仍然是急性肝衰竭（ALF）的主要原因，约占病例的15%。
在本研究中，我们通过将成人原代人肝细胞接种到气液界面（ALI）条件下的细胞培养插入物上，进行延长培养期，构建了一种新型的人三维（3D）肝组织模型。使用跨上皮电阻（TEER）测量对工程组织的屏障完整性进行了全面表征，并通过苏木精和伊红（H&E）染色及免疫组织化学评估了组织形态和结构。通过定量PCR（qPCR）评估药物转运体和药物代谢酶的表达水平。通过与传统的二维（2D）单层肝细胞培养物和肝球体进行比较，证明了该组织模型用于药物毒性评估的功能。为了评估该模型与DILI研究的相关性，我们将3D肝组织暴露于在人类中有充分记录的肝毒性特征的化合物中。通过量化释放到培养基中的生物标志物如丙氨酸氨基转移酶（ALT）和天冬氨酸氨基转移酶（AST）来监测肝功能。
工程化的3D肝组织模型呈现出明显的顶端和基底外侧表面，反映出一种极化和分层的结构，与天然肝组织非常相似。形态学和表型分析证实了该组织的器官样特征。基因表达谱显示参与药物转运、代谢和清除的肝特异性基因水平升高。在功能上，该组织将咪达唑仑（细胞色素P450 3A4（CYP3A4）酶的底物）代谢为其主要代谢产物1-羟基咪达唑仑。在反复暴露于非阿尿苷（一种已停用的抗乙肝药物，已知在人类中会引起严重肝毒性）后，该组织模型表现出屏障受损、白蛋白产生减少以及ALT和AST水平以时间和浓度依赖性方式升高。
结果强烈表明该模型在预测人类药物反应方面的生理相关性和功能。因此，这种可以培养数周并以半高通量形式生产的工程化3D器官样人肝组织模型为在体外微环境中研究药物性肝毒性创造了机会。重建的3D肝组织模型可作为旨在减少实验中动物使用的替代方法的工具。
