High‐Throughput Assessment of Mechanistic Toxicity of Chemicals in Miniaturized 3D Cell Culture

Abstract

High-content imaging (HCI) assays on two-dimensional (2D) cell cultures often do not represent in vivo characteristics accurately, thus reducing the predictability of drug toxicity/efficacy in vivo. On the other hand, conventional 3D cell cultures are relatively low throughput and possess difficulty in cell imaging. To address these limitations, a miniaturized 3D cell culture has been developed on a micropillar/microwell chip platform with human cells encapsulated in biomimetic hydrogels. Model compounds are used to validate human cell microarrays for high-throughput assessment of mechanistic toxicity. Main mechanisms of toxicity of compounds can be investigated by analyzing multiple parameters such as DNA damage, mitochondrial impairment, intracellular glutathione level, and cell membrane integrity. IC 50 values of these parameters can be determined and compared for the compounds to investigate the main mechanism of toxicity. This paper describes miniaturized HCI assays on 3D-cultured cell microarrays for high-throughput assessment of mechanistic profiles of compound-induced toxicity. C 2018 by John Wiley & Sons, Inc.Keywords: 3D cell culture r assay miniaturization r high-content imaging r mechanistic toxicity r microarray chip platform -Y (2019).High-throughput assessment of mechanistic toxicity of chemicals in miniaturized 3D cell culture.This protocol provides detailed steps involved in high-throughput cell printing with a bioprinter (S+ Microarrayer) for the generation of miniaturized 3D cell culture on a micropillar/microwell chip platform and the measurement of dose responses of model compounds in the miniaturized 3D culture of human cells using an automated fluorescent microscope (S+ Scanner) for HCI assays. Incorporating 3D cell growth on the chip platform in combination with HCI assays can be established as a reliable technique for testing toxicity of compounds in an early stage of preclinical drug discovery and better predict in vivo toxicity in clinical trials. The procedures provided below enable users to efficiently determine the mechanism of toxicity of unknown compounds in high-throughput and without the excessive use of cells, hydrogels, and other reagents. MaterialsHep3B human hepatoma cell line (ATCC# HB-8064, RRID:CVCL_0326) as a model cell line Complete RPMI-1640 medium for culturing Hep3B cells (see recipe) 1× phosphate-buffered saline (PBS; see recipe)Joshi et al.

Digital light processing-based bioprinting of microtissue hydrogel arrays using dextran-induced aqueous emulsion ink

“…46 It is conducive to high-throughput and multiple analysis of various cell-based detection and application and can significantly reduce the amount of analysis. 47 The previous study proved that the GelMA/ dextran porous bio-inks could be printed with high precision and benefit cell survival. As mentioned above, the cell migration rate was the highest when the ratio of GelMA/ dextran was 2: 1, so we selected this bio-ink ratio for printing and analysis during microtissue array printing.…”

Multiparametric nanoparticle-induced toxicity readouts with single cell resolution in HepG2 multicellular tumour spheroids

“…Using Hoechst 33342, TMRM and calcein AM, Joshi and colleagues quantitatively evaluated mitochondrial membrane potential, oxidative stress and cell membrane integrity at the spheroid level. 40 We selected propidium iodide (PI) to measure late stages of cell death, a dye that is comparatively well-established in flow cytometry cytotoxicity studies and is considered highly specific. 41 PI was also used recently to detect toxicity in HepG2 spheroids, specifically incubated with silver oxide and zinc oxide NPs.…”

Current Perspectives on Gastrointestinal Models to Assess Probiotic-Pathogen Interactions

“…These cell lines can be passaged indefinitely and have been utilized extensively to perform preliminary screening and mechanistic studies. In addition, high throughput screening and imaging techniques are available, although in vivo characteristics may not be represented accurately ( Joshi et al, 2019 ). It is the simplicity and efficiency of 2D cell lines that have made them so popular for in vitro studies ( Bédard et al, 2020 ).…”