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High-throughput multiplex assays with mouse macrophages on pillar plate platforms
Parnian Bigdelou1, Ka Keung Chan2, Jinshan Tang3
1Department of Chemical & Biomedical Engineering, Cleveland State University, Cleveland, OH, 44115, USA.
Insights
Researchers developed a 3D micropillar platform for high-throughput immune cell analysis. This new method accurately measures macrophage responses, identifying a specific N-glycan polymer with significant immune modulation activity.
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.
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.
