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

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.