Investigation On Cell Synchronous Calcium Oscillations Based On Microfluidic Chip

Ca²⁺, a critical important second messenger in cell signal transductions, regulates almost all physiological activities. Calcium oscillations are a universal signalling mode of cytosolic free Ca²⁺ in both excitable and non-excitable cells. The features of calcium oscillations, frequency, amplitude and phase, encode the specificity of calcium signalling. The amplitude and frequency of calcium oscillations triggered through surface receptors vary among individual cells over time, resulting in a mixture of stimulus waveforms that complicates analysis. Thus,it's beneficial to the research of the potential encoding mechanism of calcium signalling using controllable chemical stimulation of cells to generate synchronous calcium oscillations.PDMS-based microfluidic chips are advantageous for observation and detection, due to reasons such as transparency, highly designable chip patterns, comparable channel sizes to cells, controllable fluid flow and potential for integration and automation. All these traits make microfluidic chips suitable for the investigation of the intracellular Ca²⁺ signaling upon chemical stimulation. Here, microfluidic chips with Y-shaped microchannels are designed and fabricated. After optimizing the method to switch solutions rapidly, HeLa cells are cultured within the microchannels, treated with thapsigargin, and then stimulated with different concentrations of extracellular Ca²⁺ through switching solutions rapidly. Intracellular free Ca²⁺ signal is detected at the same time. As a result, HeLa cells were artificially induced to generate synchronous calcium oscillations, producing a uniform frequency across the cell population with a relatively constant amplitude. In addition, the amplitude of the oscillations increased with increasing concentration of extracellular Ca²⁺.In summary, the developed microfluidic method is suitable for investigating the intracellular Ca²⁺ signaling upon various chemical stimulations. For example, adenylyl cyclases catalyze ATP to turn to cyclic adenosine monophosphate (cAMP) in many signal pathways, and their activity can be regulated by the concentration of intracellular free Ca²⁺. Thus, on-chip detection of cAMP in cells with synchronous calcium oscillations can further reveal the interaction between the two second messengers, Ca²⁺ and cAMP within intracellular signaling pathways.