Conventional drug screening processes are a time-consuming and expensive endeavor, but highly rewarding when they are successful. To identify promising lead compounds, millions of compounds are traditionally screened against therapeutic targets on human cells grown on the surface of 96-wells. These two-dimensional (2D) cell monolayers are physiologically irrelevant, thus, often providing false-positive or false-negative results, when compared to cells grown in three-dimensional (3D) structures such as hydrogel droplets. However, 3D cell culture systems are not easily amenable to high-throughput screening (HTS), thus inherently low throughput, and requiring relatively large volume for cell-based assays. In addition, it is difficult to control cellular microenvironments and hard to obtain reliable cell images due to focus position and transparency issues. To overcome these problems, miniaturized 3D cell cultures in hydrogels were developed via cell printing techniques where cell spots in hydrogels can be arrayed on the surface of glass slides or plastic chips by microarray spotters and cultured in growth media to form cells encapsulated 3D droplets for various cell-based assays. These approaches can dramatically reduce assay volume, provide accurate control over cellular microenvironments, and allow us to obtain clear 3D cell images for high-content imaging (HCI). In this review, several hydrogels that are compatible to microarray printing robots are discussed for miniaturized 3D cell cultures.

The majority of high-content imaging (HCI) assays have been performed on two-dimensional (2D) cell monolayers for its convenience and throughput. However, 2D-cultured cell models often do not represent the in vivo characteristics accurately and therefore reduce the predictability of drug toxicity/efficacy in vivo. Recently, three-dimensional (3D) cell-based HCI assays have been demonstrated to improve predictability, but its use is limited due to difficulty in maneuverability and low throughput in cell imaging. To alleviate these issues, we have developed miniaturized 3D cell culture on a micropillar/microwell chip and demonstrated high-throughput HCI assays for mechanistic toxicity. Briefly, Hep3B human hepatoma cell line was encapsulated in a mixture of alginate and fibrin gel on the micropillar chip, cultured in 3D, and exposed to six model compounds in the microwell chip for rapidly assessing mechanistic hepatotoxicity. Several toxicity parameters, including DNA damage, mitochondrial impairment, intracellular glutathione level, and cell membrane integrity were measured on the chip, and the IC values of the compounds at different readouts were determined to investigate the mechanism of toxicity. Overall, the Z' factors were between 0.6 and 0.8 for the HCI assays, and the coefficient of variation (CV) were below 20%. These results indicate high robustness and reproducibility of the HCI assays established on the miniaturized 3D cell culture chip. In addition, it was possible to determine the predominant mechanism of toxicity using the 3D HCI assays. Therefore, our miniaturized 3D cell culture coupled with HCI assays has great potential for high-throughput screening (HTS) of compounds and mechanistic toxicity profiling.

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.

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