High-content imaging assays on a miniaturized 3D cell culture platform

Abstract

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.

An adaptable soft-mold embossing process for fabricating optically-accessible, microfeature-based culture systems and application toward liver stage antimalarial compound testing

“…As such, efforts have focused on engineering platforms that conform to the standards of commercial microtiter plates while maintaining critical features necessary for sophisticated organ-mimetic in vitro models. 2,3 Achieving broad applicability of such in vitro model platforms requires fabrication strategies that are compatible with a variety of designs, each having specific features relevant to the organ of interest, and some degree of scalability for use in multiple research settings. 4 The human liver is the largest internal organ, performs over 500 physiological functions, including metabolism of most xenobiotics, and is subject to a variety of disease states, including those caused by cytotoxic side-effects of xenobiotics and infectious diseases.…”

Biophysical and biomechanical properties of neural progenitor cells as indicators of developmental neurotoxicity

“…Images of cells in individual wells were acquired using an automated fluorescent microscope (S + scanner, Samsung Eletro-Mechanics, Co. (SEMCO), South Korea). (Details in supplementary methods) (Joshi et al 2018b).…”

“…After 24 h of compound exposure, surviving cells (n = 4 wells/concentration/compound) were stained with 15 µM of Hoechst 33342 (Thermo Fisher Scientific; Ex-352 nm/ Em-416 nm) in 1 × PBS (pH ~ 7.2) for 30 min to assess changes in nucleic DNA content. Nuclei were imaged using the S + scanner with a 4 × objective and a blue fluorescence filter from Semrock (DAPI-5060C-000) (Joshi et al 2018b).…”

“…Mitochondrial impairment, i.e., changes in mitochondrial membrane potential, in the presence of the compounds was assessed using tetramethyl rhodamine methyl ester (TMRM; Thermo Fisher Scientific; Ex-545 nm/Em-600 nm). The cells were stained with 0.5 µM of TMRM after 24 h of compound exposure and imaged using the S + scanner with a 4 × objective and an orange filter from Semrock (TxRed-4040C-000) (Joshi et al 2018b).…”

“…Lower GSH levels are associated with increased concentrations of glutathione disulfide (GSSG) leading to a decrease in GSH/GSSG ratio, a critical indicator of cellular health. The cells were imaged at 4 × magnification using the S + scanner with the blue filter (Joshi et al 2018b).…”