An Innovative Method of Endosome Isolation from Mouse Heart Tissues and Co-Culture with Cardiomyocytes

Endosomes are membrane-bound organelles playing essential roles in intracellular trafficking, signal transduction, and membrane recycling. Although their functions in cardiovascular biology are gaining recognition, efficient isolation of endosomes from cardiac tissue remains a significant technical challenge. We developed a rapid and high-yield protocol for isolating endosomes from mouse heart tissue by combining subcellular fractionation with an ultrafast nanofiltration isolation platform. This method includes sequential centrifugation, nanofiltration, and nanoporous membrane-based retrieval of vesicles. Isolated endosomes were characterized by western blotting, dynamic light scattering (DLS), and nano-flow cytometry (NanoFCM) analysis. To assess the bioactivity, purified endosomes were co-cultured with primary neonatal cardiomyocytes. The endosomal markers EEA1 and Rab7 were highly enriched in isolated vesicles. DLS analysis revealed a mean vesicle diameter of 187.30 ± 23.42 nm and a zeta potential of -26.60 ± 5.79 mV (n = 12). NanoFCM quantification yielded approximately 1.07 ± 0.31 × 10¹² particles/mL (n = 12) from 100 mg of heart tissue. Functional uptake of endosomes by cardiomyocytes was observed following in vitro co-culture, indicating preservation of vesicle integrity and activity. This integrated isolation platform provides a reproducible and efficient method for the extraction of high-purity endosomes from cardiac tissues. The protocol facilitates downstream biochemical and functional studies, offering a valuable tool for investigating the roles of endosomes in cardiovascular physiology and disease.