Rapid and efficient isolation of mitochondria for assessment of mitochondrial function and transplantation

Background Isolating mitochondria for clinical use remains challenging, as traditional isolation techniques often compromise organelle integrity. To address this, we developed a rapid, gentle isolation method that maximizes yield while maintaining mitochondrial integrity. Methods We established Gentle-Mito, a two-stage workflow for efficient mitochondrial isolation. Following gentle lysis, positively charged aluminum species that adsorb to the negatively charged outer mitochondrial membrane, thereby neutralizing surface charge and inducing mitochondrial aggregation for low-speed sedimentation. The structural and functional integrity of isolated mitochondria was evaluated using biochemical and imaging-based assays. Biocompatibility was assessed through mitochondrial transplantation experiments. Outcome measures included mitochondrial yield, morphology, enzymatic activity, calcium responsiveness, mitochondrial DNA integrity, cellular internalization, cytotoxicity, and restoration of metabolic function. Results The Gentle-Mito method enables mitochondrial isolation within 30 min and achieves more than twofold higher recovery than conventional methods while preserving organelle integrity. Isolated mitochondria retained intact morphology, preserved Citrate Synthase activity, and exhibited robust calcium-induced swelling. The method also supported the isolation of mitochondrial DNA and enabled mitochondrial transplantation. Transplanted mitochondria were efficiently internalized by recipient cells, improved gap closure, remained functionally active for more than 48 h, and elicited no detectable cytotoxicity. Transplantation of isolated mitochondria restored metabolic activity and re-sensitized Osimertinib-resistant lung cancer cells. Conclusion The Gentle-Mito method provides a simple, rapid, scalable, and biocompatible platform for the isolation of intact mitochondria. By improving both mitochondrial recovery and quality, this approach may broaden the utility of mitochondrial isolation for basic research, translational studies, and the development of mitochondria-based therapeutic strategies.