Microfluidic selection and retention of a single cardiac myocyte, on-chip dye loading, cell contraction by chemical stimulation, and quantitative fluorescent analysis of intracellular calcium

A microfluidic method to study the contraction of a single cardiac myocyte (heart muscle cell) has been developed. This method integrates various single-cell operations as well as on-chip dye loading, and quantitative analysis of intracellular calcium concentration, [Ca2+](i). After the channel enlargement by on-chip etching to accommodate large-sized cardiac myoytes, a single cell is selected and retained at a V-shaped cell retention structure within the microchip. Owing to the fragile property of the cardiac myocytes that could easily be damaged by centrifugation, the calcium-sensitive fluorescent dye was loaded in the cell by on-chip dye loading. This on-chip method minimized the damage to the cells from the use of a centrifuge in the conventional method and provided a way of cellular analysis of fragile cells. Subsequently, quantitative analysis of [Ca2+](i) of a single cardiac myocyte by fluorescence measurement was achieved for the first time in a microfluidic chip, thanks to the intracellular calcium stimulant of ionomycin. The resting [Ca2+](i) of the cardiomyocyte determined was consistent with the literature value. From the spontaneous contraction study, it was found that fluorescence intensity cannot represent the [Ca2+](i) variation accurately, which implied the importance of the quantitative analysis of [Ca2+],.