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High-throughput multiplex assays with mouse macrophages on pillar plate platforms.

ABSTRACT: It is challenging to rapidly identify immune responses that reflect the state and capability of immune cells due to complex heterogeneity of immune cells and their plasticity to pathogens and modulating molecules. Thus, high-throughput and easy-to-use cell culture and analysis platforms are highly desired for characterizing complex immune responses and elucidating their underlying mechanisms as well. In response to this need, we have developed a micropillar chip and a 384-pillar plate, printed mouse macrophage, RAW 264.7 cell line in alginate on the pillar plate platforms, and established multiplex cell-based assays to rapidly measure cell viability, expression of cell surface markers, and secretion of cytokines upon stimulation with model compound, lipopolysaccharide (LPS), as well as synthetic N-glycan polymers that mimic native glycoconjugates and could bind to lectin receptors on RAW 264.7 cells. Interestingly, changes in RAW 264.7 cell viability, expression levels of cell surface makers, and release of cytokines measured from the pillar plate platforms in the presence and absence of LPS were well correlated with those obtained from their counterpart, the 96-well plate with 2D-cultured macrophages. With this approach, we identified that α2,3-linked N-sialyllactose polymer has significant macrophage modulation activity among the N-glycan polymers tested. Therefore, we successfully demonstrated that our pillar plate platforms with 3D-cultured macrophages can streamline immune cell imaging and analysis in high throughput in response to compound stimulation. We envision that the pillar plate platforms could potentially be used for rapid characterization of immune cell responses and for screening immune cell-modulating molecules.

SUBMITTER: Bigdelou P

PROVIDER: S-EPMC7572780 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Assessing the neurotoxicity of test chemicals has typically been performed using two-dimensionally (2D)-cultured neuronal cell monolayers and animal models. The in vitro 2D cell models are simple and straightforward compared to animal models, which have the disadvantage of being relatively low throughput, expensive, and time consuming. Despite their extensive use in this area of neurotoxicology research, both models often do not accurately recapitulate human outcomes. To bridge this gap and attempt to better replicate what happens in vivo, three-dimensionally (3D) cultured neural stem cells (NSCs) encapsulated in hydrogels on a 384-pillar plate have been developed via miniature 3D bioprinting. This technology allows users to print NSCs on a pillar plate for rapid 3D cell culture as well as high-throughput compound screening. For this, the 384-pillar plate with bioprinted NSCs is sandwiched with a standard 384-well plate with growth medium for 3D culture, allowing researchers to expose the cells to test compounds and stain them with various fluorescent dyes for a suite of high-content imaging assays, including assays for DNA damage, mitochondrial impairment, cell membrane integrity, intracellular glutathione levels, and apoptosis. After acquiring cell images from an automated fluorescence microscope and extracting fluorescence intensities, researchers can obtain the IC50 value of each compound to evaluate critical parameters in neurotoxicity. Here, we provide a detailed description of protocols for cell printing on a 384-pillar plate, 3D NSC culture, compound testing, 3D cell staining, and image acquisition and analysis, which altogether will allow researchers to investigate mechanisms of compound neurotoxicity with 3D-cultured NSCs in a high-throughput manner. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Three-dimensional neural stem cell culture on a 384-pillar plate Basic Protocol 2: Compound treatment and cell staining Basic Protocol 3: Image acquisition, processing, and data analysis.

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High-throughput multiplex assays with mouse macrophages on pillar plate platforms.

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