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

Area of Science:

• Immunology
• Biotechnology
• Materials Science

Background:

• Characterizing complex immune responses is challenging due to immune cell heterogeneity and plasticity.
• High-throughput, user-friendly platforms are needed for immune cell analysis and mechanism elucidation.

Purpose of the Study:

• To develop and validate a micropillar chip and 384-pillar plate platform for high-throughput 3D cell culture and multiplexed immune response assays.
• To assess the platform's ability to measure macrophage viability, cell surface marker expression, and cytokine secretion.
• To identify specific immune-modulating molecules using the developed platform.

Main Methods:

• Developed a micropillar chip and 384-pillar plate for 3D cell culture.
• Printed RAW 264.7 macrophage cell line in alginate on the pillar plate platforms.
• Established multiplex cell-based assays to measure cell viability, cell surface marker expression, and cytokine secretion.
• Stimulated cells with lipopolysaccharide (LPS) and synthetic N-glycan polymers.
• Compared results with traditional 2D-cultured macrophages in 96-well plates.

Main Results:

• The micropillar platform demonstrated high correlation with 96-well plate assays for measuring macrophage responses.
• Identified α2,3-linked N-sialyllactose polymer as having significant macrophage modulation activity among tested N-glycan polymers.
• Successfully streamlined high-throughput immune cell imaging and analysis in response to compound stimulation.

Conclusions:

• The developed 3D micropillar plate platform enables rapid, high-throughput characterization of immune cell responses.
• This platform can be utilized for screening immune cell-modulating molecules.
• The technology offers a streamlined approach for understanding complex immune cell behaviors and mechanisms.