Abstract
Cell models are becoming more complex to better mimic the in vivo environment and provide greater predictivity for compound efficacy and toxicity. There is an increasing interest in exploring the use of three-dimensional (3D) spheroids for modeling developmental and tissue biology with the goal of accelerating translational research in these areas. Accordingly, the development of high-throughput quantitative assays using 3D cultures is an active area of investigation. In this study, we have developed and optimized methods for the formation of 3D liver spheroids derived from human iPS cells and used those for toxicity assessment. We used confocal imaging and 3D image analysis to characterize cellular information from a 3D matrix to enable a multi-parametric comparison of different spheroid phenotypes. The assay enables characterization of compound toxicities by spheroid size (volume) and shape, cell number and spatial distribution, nuclear characterization, number and distribution of cells expressing viability, apoptosis, mitochondrial potential, and viability marker intensities. In addition, changes in the content of live, dead, and apoptotic cells as a consequence of compound exposure were characterized. We tested 48 compounds and compared induced pluripotent stem cell (iPSC)-derived hepatocytes and HepG2 cells in both two-dimensional (2D) and 3D cultures. We observed significant differences in the pharmacological effects of compounds across the two cell types and between the different culture conditions. Our results indicate that a phenotypic assay using 3D model systems formed with human iPSC-derived hepatocytes is suitable for high-throughput screening and can be used for hepatotoxicity assessment in vitro.
Highlights
Liver injury caused by toxicity of pharmaceutical drugs and environmental chemicals is a subject of great concern
The goal of this study was to develop and characterize confocal high-content imaging in combination with 3D image analysis methods that would be suitable for the highthroughput compound screening using liver spheroids made from induced pluripotent stem cell (iPSC)-derived hepatocytes
Human iPSC-derived hepatocytes were cultured for 7 days in a 2D format before detaching the cells, supplementing the cell suspension with extracellular matrix, and plating the mixture into ultra-low attachment (ULA) plates to initiate 3D spheroid formation
Summary
Liver injury caused by toxicity of pharmaceutical drugs and environmental chemicals is a subject of great concern. Development of new complex systems that would allow effective testing for potential liver toxicity is an area of active investigation.[1,2,3] There is a growing interest in using three-dimensional (3D) models for studying complex biology and tissue engineering. 3D models have been employed for developmental biology research and toxicity screening.[4,5,6,7,8,9] Numerous studies have reported more liver-specific functional activity in hepatocyte 3D cultures than in conventional two-dimensional (2D) monolayers,[5,10,11,12] since mechanical stress mediated by adhesion to extracellular matrix or other cells alters signal transduction and gene transcription in a variety of cell types. There has been significant progress in the development of 3D cell models and techniques during the past several years
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