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 ...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 locationdependent 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.展开更多
基金supported by The National Institute of General Medical Sciences of the National Institutes of Health under award number R35GM133610(CNJ)and R35GM147094(MDB)by The UT Dallas Seed Program for Interdisciplinary Research(SPIRe)program(MDB&CNJ)by The National Science Foundation under CBET award number 2300340.
文摘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 locationdependent 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.
