Phenotypic Characterization of Toxic Compound Effects on Liver Spheroids Derived from iPSC Using Confocal Imaging and Three-Dimensional Image Analysis

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.

Designing a blueprint for next‐generation stem cell bioprocessing development

“…Since numerous signaling pathways that sense the cellular state and phenotypic properties of single cells within their microenvironments exist, we expect an increase in future efforts toward the development of tools that detect and quantify heterogeneity. The existing evaluation methods employed for such analyses include polychromatic flow cytometry, mass cytometry, single‐cell RNA sequencing, single‐cell RT‐qPCR, microtools, and high‐resolution imaging (Avior, Biancotti, & Benvenisty, ; Morris & Daley, ; Patel et al, ; Sena, Galotto, Devitt, Connick, & Jacobi, ; Sirenko et al, ; Stern et al, ; Tsankov et al, ). Additionally, the establishment of standardized terminology, methods, and metrics will be essential for routinely extracting and communicating insights regarding biological heterogeneity.…”

“…A computer-controlled bioreactor also ensures that culture conditions are created that facilitate media exchange and creates a proper culture environment to enable large-scale growth of cells in vitro. Furthermore, these systems must be integrated with techniques and tools for measuring, monitoring, imaging, analyzing, and forecasting simulations, along with required product quality assessments (Bergholt, Albro, & Stevens, 2017;Kino-oka & Prenosil, 2000;Lim et al, 2007;Lin, Fallahi-Sichani, & Sorger, 2015;Marklein, Lam, Guvendiren, Sung, & Bauer, 2018;Nederlof, Watanabe, Burnip, Taylor, & Critchley-Thorne, 2011;Sirenko et al, 2016;Thomas et al, 2008). Integrated bioprocess designs will ultimately facilitate bioprocessing control and generate optimal levels of precision, reliability, and product quality for clinical and industrial applications.…”

Organoid technology for brain and therapeutics research

“…Organoids also have potential applications in testing efficacy and toxicity of drugs and compounds . More recently, brain organoids derived from human PSCs were performed to predicting neural toxicity.…”

“…Organoids also have potential applications in testing efficacy and toxicity of drugs and compounds. [31][32][33] More recently, brain organoids derived from human PSCs were performed to predicting neural toxicity. The brain organoids were treated with 34 toxic and 26 nontoxic chemicals and able to accurately predict 90% of neurotoxic compounds with the use of RNA sequencing and machine learning.…”

Neural organoids for disease phenotyping, drug screening and developmental biology studies

“…hPSC-derived cell types are becoming increasingly utilized as a source of material for microphysiological systems, or “organs-on-a-chip”, demonstrating that they are well positioned to serve as an improved method to asses drug efficacy and safety (Fabre, Livingston, & Tagle, 2014; Judson et al, 2014; Shinde, Sureshkumar, Sotiriadou, Hescheler, & Sachinidis, 2016; Sirenko et al, 2016). Focusing on the ability of organoids to refine toxicology testing, neural constructs engineered from multiple cell types were treated with a set of compounds, comprising both known neurotoxic compounds and control chemicals; RNA-sequencing combined with machine learning was able to accurately predict neurotoxic compounds in 90% of tests (Schwartz et al, 2015).…”