A Microsystem-based Approach for Monitoring Calcium Dynamics in Microglia during Inflammation

Introduction:: Microglia are central nervous system (CNS)-specific immune cells that respond to both immunological (cytokines) and neuronal (ATP) signals. Among these mechanisms, calcium dynamics have been shown to play a key role in microglia functions, including chemotaxis and cytokine release, but there is controversy regarding the presence of spontaneous calcium dynamics in microglia in vitro. However, current calcium dynamics studies are done using a perfusion system that takes a long time to set up and does not involve the creation of a concentration gradient during the delivery of the treatment to the cells, which can be a determining factor while imaging calcium dynamics. Therefore, there is a need for new tools that are both efficient and provide a concentration gradient. To address this issue, we introduce a Calcium Monitoring Micro-Total Analysis System (CAM-μTAS) using microfluidics, which provides efficient and controlled delivery of treatments to cells following a concentration gradient. This system combines the integrated pumping capabilities of lifting gate microvalves with the gradient monitoring capabilities of quake valves, enabling efficient changes in cell culture media, calcium sensor incubation, and cell exposure to cytokines. The CAM-μTAS chip also includes a microfluidic flow rectifier that allows for zero backflow. With this system, calcium imaging of microglia during a dosed treatment of a cytokine (IL-6, TNF-α ) and ATP mixture that modeled neuroinflammation was performed and optimized.


Materials and Methods:: The calcium monitoring micro-total analysis system (CAM-μTAS) consisted of three layers: a fluidic layer with microchannel to allow fluid transfer, a pneumatic control layer, and a featureless glass wafer. Using standard photolithography and soft-lithography techniques, customized microchannels and valves were fabricated and fused to transparent polydimethylsiloxane (PDMS) substrates. The different layers were strategically positioned to form an array of microvalves used as micropump to allow fluid transfer. To demonstrate the utility of the CAM-μTAS, the PDMS chip was functionalized for cell adhesion by a treatment of poly-L-lysine [0.01%] and fibronectin [11μg/mL]. After washing out the solution, microglia cells suspended in media were injected in the cell chamber and cultured for 24 hours to allow cells to adhere to the PDMS surface. Media change was performed by actuating a series of adjacent microvalves to create micropumps using a custom-made solenoid valve control system and LabVIEW software. In the adjacent chamber, a solution of IL-6 [10ng/mL], TNF-α [5ng/mL], and ATP [200μM] was added. Finally, the chamber separating valve was opened for the cytokines to reach the cells following a concentration gradient, and the calcium dynamics was imaged and recorded every 50ms for 3min. using a fluorescent microscope.

Results, Conclusions, and Discussions:: As illustrated in Fig1A, the CAM-μTAS fabricated to incorporate both the lifting gate valves and the quake valve. The setting and orientation of the valves on the chip allowed to culture cells without any human interactions. The custom-made LabVIEW software ensured full automation. It only took six seconds to perform media changes which is more efficient than the traditional media replacement methods which can take several minutes. As shown in Fig 1B, a concentration gradient was generated on the chip to treat the cells with cytokines. The treatment was delivered to the cells after 2:12 minutes. Finally, microglia calcein-Am [2μM] was loaded on the chip and calcium level images of the cells were recorded which validated the utility of the CAM-μTAS (Fig 1C). After the treatment reached the cells, a response was recorded by the change in fluorescence as shown in Fig 1E. These responses yielded an average amplitude 7 times the baseline as shown in Fig 1D.



Conclusion: A fully automated microfluidics device has been developed enabling the measurement of calcium dynamics in microglia following a cytokine treatment. Equipped with an automated fluid pump, and chamber separating normally opened valve, this device enables programmed and metered fluidic manipulations with no human interaction. We were able to quantify a 7-fold increase in calcium signaling in a gradient of inflammatory signals. Future work on this technology will involve culturing different types of cells in the different chambers and evaluating their interactions, including calcium dynamics, migration, and cell-cell interactions.


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