Research On Cellular Calcium Signaling By Microfluidic Chips

Ca²⁺ is a universal intracellular messenger. Spatiotemporal variation of intracellular Ca²⁺ concentration and its related signal transduction lead to the regulation of gene expression so as to affect or even determine cellular behaviors. Thus, researches on intracellular calcium signaling and the propagation of intercellular calcium signaling play an important role in discovering mechanisms of numerous physiological processes and overcoming human diseases.Microfluidic chip has been a new platform for cell research. This paper utilized microfluidic chips for investigating cellular calcium signaling and achieved creative results as follows:(1) We developed a local dosing system based on microfluidic multiple laminar flows for chemically initiating intercellular calcium signaling known as calcium wave. Cells were introduced into a ?Y?-shaped microchannel by using negative pressure pulses, and located at the target area. After cell adhesion, cells were incubated with Ca²⁺ fluorescence probe. According to the distance between the target cell and the channel wall, the flow rates of the micro syringe pumps were set. Adenosine triphosphate (ATP) and buffer solutions were driven into the microchannel by positive pressures, forming two parallel laminar flows. Part of the target cell was exposed to ATP flow and the adjacent cells were exposed to the buffer flow. The propagation of the calcium wave from the target cell to adjacent cells was observed. The direction of this calcium wave was opposite to the flow direction, indicating that this calcium wave was not mediated by the diffusion of ATP that released from the target cell to adjacent cells. After the addition of octanol into the buffer for inhibition of gap junctions, the calcium wave vanished, suggesting that this calcium wave was mainly mediated by gap junctions.(2) We realized millisecond chemical stimulation of single cells by using two negative pressure systems. A single cell was seeded into the target area of a cross-shaped microchannel. By controlling two negative pressures on two channel outlets, the status of ATP and buffer fluids in the microchannel switched between hydrodynamic gating and hydrodynamic focusing, leading to the rapid solution exchange (17 ms) upon the target cell and the generation of a millisecond ATP pulse that passed by the cell. Results suggested that 100 ms stimulation of single cells with 20μM ATP minimized the desensitization of cell-surface receptors and consequently the receptors could recover from desensitization completely within 50 s. Thus, control assays could be done on the same single cells. The amplitude of calcium spikes induced by 20μM ATP with increasing durations from 50 ms to 100 ms and to 1 s maintained at a similar level. As the concentration of 100 ms ATP pulses increased from 2μM to 20μM and to 200μM, the amplitude of resulting calcium spikes elevated. Both results rigorously proved that ATP-induced calcium release is concentration dependent. However, when the cell was exposed to 2μM ATP pulses with increasing durations from 30 ms to 50 ms and to 100 ms, the amplitude of calcium spikes elevated, suggesting a time dependent calcium release.(3) We realized spatiotemporal control of chemical microenvironment of the cell with two negative pressure systems and developed a new approach for investigating intercellular signaling. Soon after the switch between hydrodynamic gating and focusing, the solution exchange around the target cell could be completed in ¹00 ms. During hydrodynamic focusing, the distance of transverse diffusion of ATP was ²5μm. Thus, time-controllable local dosing to a single cell was conducted, inducing calcium waves.