Ischemia/reperfusion injury of primary porcine cardiomyocytes in a low-shear microfluidic culture and analysis device

Ischemia/reperfusion (I/R) injury was induced in primary porcine cardiomyocytes in a low-shear microfluidic culture chip. The chip was capable of sustaining the cardiomyocyte culture and inducing I/R injury by subjecting the cells to periods of hypoxia lasting 3-4 hours followed by normoxia. Mitochondrial membrane potential was assayed using MitoTracker Red to follow mitochondrial depolarization, the earliest stage of apoptosis. Cell adhesion and morphology were also determined simultaneously with fluorescence measurements. Changes in membrane potential were observed earlier than previously reported, with mitochondria becoming depolarized as early as 2 hours into the ischemia period. The cells with depolarized mitochondria were deemed apoptotic. Out of 38-61 cells per time frame, the fraction of apoptotic cells was found to be similar to control samples (3%) at two hours of ischemia, which increased up to 22% at the end of the ischemia period as compared to 0% in the control samples. Morphological analysis of cells showed that 4 hours of ischemia followed by reperfusion produced blebbing cells within 2 hours of restoring oxygen to the chip. This approach is a versatile method for cardiomyocyte stress, and in future work additional analytical probes can be incorporated for a multi-analyte assay of cardiomyocyte apoptosis.