Three Dimensional Hepatotoxicity Screening using Corning® HepatoCells, the Spheroid Microplate and the SCREEN-WELL® Hepatotoxicity library Presenter: Ute Vespermann Hilary Sherman, Hannah J. Gitschier, David H. Randle Corning Incorporated, Life Sciences, Kennebunk, Maine USA Warranty/Disclaimer: Unless otherwise specified, all products are for research use only. Not intended for use in diagnostic or therapeutic procedures. Not for use in humans. Corning Life Sciences makes no claims regarding the performance of these products for clinical or diagnostic applications Corning is a registered trademark of Corning Incorporated, One Riverfront Plaza, Corning, NY 14831-0001 All other trademarks included in the document are the property of their respective owners © 2016 Corning Incorporated Abstract Summary/Conclusions Having the right model for drug screening is essential for predicting compounds that may cause drug-induced liver injury. Three dimensional (3D) models offer significant improvements over traditional two dimensional monolayer cell culture in terms of maintaining morphological and functional characteristics of tissue, and may provide a better representation of in vitro drug toxicity1. Here we demonstrate how Corning HepatoCells, an immortalized alternative to primary human hepatocytes, in conjunction with Corning Spheroid Microplates can be a utilized for a 3D drug screen to discover potential hepatotoxins. Hepatospheres formed using Corning HepatoCells were compared to spheroids formed using alternative hepatocyte-like models. Cell viability, urea and albumin were measured to assess hepatotoxicity after exposure to the SCREEN-WELL Hepatotoxicity library from Enzo Life Sciences, a library consisting of 238 compounds with a variety of structurally and mechanistically different compound classes, as well as nontoxic controls. Selected hits identified in the 3D screen, which significantly reduced cell viability of the hepatospheres, were then assessed in a dose-dependent manner for potency analysis. These results demonstrate that Corning HepatoCells, together with Corning Spheroid Microplates, are powerful tools that can be used for reliable and reproducible 3D hepatotoxicity screening. • Corning spheroid microplates allow for the formation of consistent sized, single spheroids in each well, available in both 96 and 384 well formats, making them a useful tool for 3D screening. • Hepatospheres formed using the Corning spheroid microplate are amenable to histological analysis. • The opaque walls and clear, round well-bottom of the spheroid microplates allow for luminescent assays to be conducted in the plate without the need for a transfer step. • HepatoCells offer an ideal hepatic cell alternative to commonly used HepaRG cells, offering a larger assay window for luminescent ATP assays, which is amenable to screening. • HepatoCells displayed increased or equivalent sensitivity to known hepatotoxins tamoxifen, troglitazone and nicardipine upon potency analysis, compared to HepG2 and HepaRG cells. Representative photomicrographs of HepatoCells,HepG2, and HepaRG spheroids after 7 days, seeded at 6,000 cells/well, 200 cells/well, and 6,000 cells/well respectively (40x). Spheroid Size Optimization HepatoCells (Corning Cat. No. 354881), HepG2 cells (ATCC® Cat. No. HB-8065), and HepaRG™ cells (Life Technologies Cat. No. HPRGC10) were seeded at various concentrations in 384 well spheroid microplates (Corning Cat. No. 3830) to optimize for the subsequent screens. HepatoCells and HepG2 cells were seeded using 50 µL Corning Culture Medium for HepatoCells (Corning Cat. No. 354882) containing 10 % fetal bovine serum (Corning Cat. No. 35-010-CV). HepaRG cells were seeded using William’s medium (Life Technologies Cat. No. 12551-032) supplemented to 1x with HepaRG Thaw, Plate & General Purpose Medium Supplement (Life Technologies Cat. No. HPRG670) and 1x GlutaMAX (Life Technologies Cat. No. 35050-061). Medium was changed every other day using