A microfluidic platform for isolating functional mitochondria and restoring bioenergetic activity in hypoxia-injured...

Hypoxic stress is known to severely impair mitochondrial function in cardiomyocytes, leading to disruption of intracellular bioenergetic homeostasis, reduced ATP production, and compromised cellular performance. Because cardiomyocytes rely heavily on mitochondrial oxidative metabolism to meet their high energy demands, mitochondrial dysfunction represents a central mechanism underlying hypoxia-induced cardiac injury. In this study, a microfluidic-based platform was developed for mitochondrial isolation and to investigate whether the delivery of functionally preserved mitochondria could enhance mitochondrial bioenergetic function in hypoxia-injured cardiomyocytes. Human AC16 cardiomyocytes were used as the experimental model. Mitochondria were isolated using a centrifugal microfluidic disruption system operated under optimized centrifugation conditions and were directly compared with mitochondria obtained using a commercial isolation kit. Hypoxic injury was induced by culturing AC16 cells at 1% O₂ for 24 h. Mitochondrial incorporation and functional status were evaluated using MitoTracker staining, JC-1 analysis, intracellular ATP quantification, and Seahorse XF mitochondrial stress tests. Microfluidic devices can be used to extract mitochondria from healthy cells, and this method achieves similar functional results to mitochondria extracted using commercial reagents. Hypoxic cells show a significant decrease in cell membrane potential and overall respiratory capacity. Returning the extracted mitochondria to hypoxic cells shows partial recovery of mitochondrial function. Seahorse analysis reveals partial recovery of basal respiration, maximal respiration, reserve respiration, and ATP-coupled respiration, although not reaching the levels of healthy cells. This demonstrates the effectiveness of this method. These results indicate that mitochondria isolated using a microfluidic approach remain functionally competent and are capable of partially improving mitochondrial bioenergetic function in hypoxia-injured cardiomyocytes. The proposed microfluidic platform provides a controllable and reproducible strategy for mitochondrial isolation and functional evaluation and may serve as a useful tool for studying mitochondrial dysfunction and recovery under hypoxic conditions.