3D engineered In vitro hepatospheroids for studying drug toxicity and metabolism. (February 2017)

• Record Type: Journal Article
• Authors: Chitrangi, Swati; Nair, Prabha; Khanna, Aparna
• Is Part Of: Toxicology in vitro. Volume 38(2017)
• Page Start: 8
• Page End: 18
• Publication Date: 2017-02
• Subjects: 3D Three-dimensional -- 2D Two-dimensional -- CYP cytochrome P450 -- GEVAC Gelatin-Vinyl Acetate -- MSCs Mesenchymal stem cells -- hUC-MSCs human umbilical cord derived-mesenchymal stem cells -- HLCs Hepatocyte-like cells -- DMEM-LG Dulbecco's Modified Eagle's-low glucose -- FBS Fetal Bovine Serum -- CD Cluster of Differentiation -- AST aspartate aminotransferase -- ALT alanine aminotransferase -- HR-LC/MS high resolution-liquid chromatography mass spectrometry -- m/z mass-to-charge ratio -- 7-AAD 7-amino actinomycin -- DCFDA 2′, 7′-dichlorofluorescein diacetate -- ROS reactive oxygen species -- LDH lactate dehydrogenase -- APAP acetaminophen -- EtOH ethanol -- NCE new chemical entities -- ADME absorption, distribution, metabolism and excretion -- DMSO dimethyl sulfoxide -- h hour -- ELISA enzyme linked immuno sorbent assay -- FGF Fibroblast Growth Factor -- HGF Hepatocyte Growth Factor -- DEX Dexamethasone -- ITS Insulin Transferrin Selenium -- OSM Oncostatin M -- MTT 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide -- DAPI 4′, 6-diamidino-2-phenylindole -- min minute -- PHH Primary Human Hepatocytes; Mesenchymal stem cell -- Hepatospheroid -- Polymer scaffold -- Hepatotoxicity testing -- Drug metabolism -- Pharmacokinetics; Toxicity testing -- In vitro -- Periodicals; Toxicology -- Periodicals; 615.9
• DOI: 10.1016/j.tiv.2016.10.009
• Languages: English
• ISSNs: 0887-2333

Abstract

Abstract: 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 = 50–80 μm) 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 0.011 ml/h/10 6 cells and 0.021 ml/h/10 6 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. Graphical abstract: Highlights: Established stem cell-derived hepatospheroids in vitro drug screening model Comparison of 2D HLCs and 3D hepatospheroids for drug metabolism Effect of toxicity (ethanol, acetaminophen) on the functionality of hepatocytes ROS production indicated biomimetic toxic response. In vitro drug metabolite profiling indicated the presence of active CYP enzymes.