Chitrangi Swati, Nair Prabha, Khanna Aparna
Department of Biological Sciences, Sunandan Divatia School of Science, SVKM'S NMIMS (Deemed-to-be ) University, V. L Mehta road, Vile Parle (West), Mumbai 400056, Maharashtra, India.
Division of Tissue Engineering and Regeneration Technologies, Biomedical Technology Wing, Shree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram 695012, Kerala, India.
Toxicol In Vitro. 2017 Feb;38:8-18. doi: 10.1016/j.tiv.2016.10.009. Epub 2016 Oct 26.
Drug toxicity is one of the reasons for late stage drug attrition, because of hepatotoxicity. Various in vitro liver models like primary human hepatocytes, immortalized human hepatic cell lines, liver slices and microsomes have been used; but limited by viability, hepatic gene expression and function. The 3D-engineered construct of hepatocyte-like-cells (HLCs) differentiated from stem cells, may provide a limitless source of hepatocytes with improved reproducibility. Towards this end, we used hepatospheroids (diameter=50-80μm) differentiated from human-umbilical-cord-mesenchymal stem cells (hUC-MSCs) on 3D scaffold GEVAC (Gelatin-vinyl-acetate-copolymer) as in vitro model for studying drug metabolism/toxicity. Our data demonstrated that hUC-MSCs-derived-hepatospheroids cultured on GEVAC expressed significantly higher drug-metabolizing enzymes (CYPs) both at mRNA and activity level compared to 2D culture, using HR-LC/MS. We further showed that hepatospheroids convert phenacetin (by CYP1A2) and testosterone (by CYP3A4) to their human-specific metabolites acetaminophen and 6β-hydroxytestosterone with a predictive clearance rate of 0.011ml/h/10 cells and 0.021ml/h/10 cells respectively, according to first-order kinetics. Hepatotoxicity was confirmed by exposing hepatospheroids to ethanol and acetaminophen; ROS generation, cell viability, cytoskeleton structure, elevation of liver function enzymes, i.e. AST and ALT, was analyzed. To the best of our knowledge, this is the first report to use hUC-MSCs-derived-hepatospheroids on GEVAC as in vitro model for drug metabolism/toxicity study; which can replace the conventional 2D-models used in drug development.
药物毒性是药物研发后期失败的原因之一，这是由肝毒性导致的。人们已经使用了各种体外肝脏模型，如原代人肝细胞、永生化人肝细胞系、肝切片和微粒体；但这些模型受到细胞活力、肝脏基因表达和功能的限制。由干细胞分化而来的类肝细胞（HLCs）构建的3D工程结构，可能会提供具有更高可重复性的无限肝细胞来源。为此，我们使用了在3D支架GEVAC（明胶-醋酸乙烯酯共聚物）上由人脐带间充质干细胞（hUC-MSCs）分化而来的肝球（直径=50-80μm）作为体外模型来研究药物代谢/毒性。我们的数据表明，与二维培养相比，在GEVAC上培养的hUC-MSCs来源的肝球在mRNA和活性水平上均显著表达更高的药物代谢酶（CYPs），采用高分辨液相色谱/质谱法检测。我们进一步表明，根据一级动力学，肝球分别将非那西丁（通过CYP1A2）和睾酮（通过CYP3A4）转化为其人体特异性代谢产物对乙酰氨基酚和6β-羟基睾酮，预测清除率分别为0.011ml/h/10个细胞和0.021ml/h/10个细胞。通过将肝球暴露于乙醇和对乙酰氨基酚来确认肝毒性；分析了活性氧生成、细胞活力、细胞骨架结构、肝功能酶即AST和ALT的升高情况。据我们所知，这是第一份使用在GEVAC上的hUC-MSCs来源的肝球作为药物代谢/毒性研究体外模型的报告；它可以替代药物开发中使用的传统二维模型。
