RESUMO
Neuroinflammation is characterized by the elevation of cytokines and adenosine triphosphate (ATP), which in turn activates microglia. These immunoregulatory molecules typically form gradients in vivo, which significantly influence microglial behaviors such as increasing calcium signaling, migration, phagocytosis, and cytokine secretion. Quantifying microglial calcium signaling in the context of inflammation holds the potential for developing precise therapeutic strategies for neurological diseases. However, the current calcium imaging systems are technically challenging to operate, necessitate large volumes of expensive reagents and cells, and model immunoregulatory molecules as uniform concentrations, failing to accurately replicate the in vivo microenvironment. In this study, we introduce a novel calcium monitoring micro-total analysis system (CAM-µTAS) designed to quantify calcium dynamics in microglia (BV2 cells) within defined cytokine gradients. Leveraging programmable pneumatically actuated lifting gate microvalve arrays and a Quake valve, CAM-µTAS delivers cytokine gradients to microglia, mimicking neuroinflammation. Our device automates sample handling and cell culture, enabling rapid media changes in just 1.5 s, thus streamlining the experimental workflow. By analyzing BV2 calcium transient latency to peak, we demonstrate location-dependent microglial activation patterns based on cytokine and ATP gradients, offering insights contrasting those of non-gradient-based perfusion systems. By harnessing advancements in microsystem technology to quantify calcium dynamics, we can construct simplified human models of neurological disorders, unravel the intricate mechanisms of cell-cell signaling, and conduct robust evaluations of novel therapeutics.
RESUMO
Calcium dynamics significantly influence microglial cell immune responses, regulating activation, migration, phagocytosis, and cytokine release. Understanding microglial calcium signaling is vital for insights into central nervous system immune responses and their impact on neuroinflammation. We introduce a calcium monitoring micro-total analysis system (CAM-µTAS) for quantifying calcium dynamics in microglia (BV2 cells) within defined cytokine microenvironments. The CAM-µTAS leverages the high efficiency pumping capabilities of programmable pneumatically actuated lifting gate microvalve arrays and the flow blocking capabilities of the Quake valve to deliver a cytokine treatment to microglia through a concentration gradient, therefore, biomimicking microglia response to neuroinflammation. Lifting gate microvalves precisely transfer a calcium indicator and culture medium to microglia cells, while the Quake valve controls the cytokine gradient. In addition, a method is presented for the fabrication of the device to incorporate the two valve systems. By automating the sample handling and cell culture using the lifting gate valves, we could perform media changes in 1.5 seconds. BV2 calcium transient latency to peak reveals location-dependent microglia activation based on cytokine and ATP gradients, contrasting non-gradient-based widely used perfusion systems. This device streamlines cell culture and quantitative calcium analysis, addressing limitations of existing perfusion systems in terms of sample size, setup time, and biomimicry. By harnessing advancements in microsystem technology to quantify calcium dynamics, we can construct simplified human models of neurological disorders, unravel the intricate mechanisms of cell-cell signaling, and conduct robust evaluations of novel therapeutics.
