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.

由于免疫细胞复杂的异质性及其对病原体和调节分子的可塑性，快速识别反映免疫细胞状态和能力的免疫反应具有挑战性。因此，非常需要高通量且易于使用的细胞培养和分析平台来表征复杂的免疫反应并阐明其潜在机制。针对这一需求，我们开发了微柱芯片和 384 柱板，在柱板平台上打印小鼠巨噬细胞、海藻酸盐中的 RAW 264.7 细胞系，并建立了多重细胞检测方法来快速测量细胞活力、表达模型化合物、脂多糖 (LPS) 以及模拟天然糖缀合物并可与 RAW 264.7 细胞上的凝集素受体结合的合成N-聚糖聚合物刺激细胞表面标记物和细胞因子的分泌。有趣的是，在存在和不存在 LPS 的情况下，从支柱板平台测量到的 RAW 264.7 细胞活力、细胞表面标记表达水平以及细胞因子释放的变化与从对应的 2D 96 孔板获得的结果密切相关。 -培养的巨噬细胞。通过这种方法，我们发现α2,3-连接的N-唾液酸乳糖聚合物在测试的N-聚糖聚合物中具有显着的巨噬细胞调节活性。因此，我们成功证明了我们的具有 3D 培养巨噬细胞的柱板平台可以简化免疫细胞成像和高通量分析，以响应复合刺激。我们设想柱板平台有可能用于快速表征免疫细胞反应和筛选免疫细胞调节分子。