Optimized Isolation and Cryopreservation of Functional Mitochondria for Transplantation and Therapeutic Applications.

Mitochondria play a central role in numerous physiological and pathological processes, and mitochondrial transplantation is emerging as a promising strategy to restore cellular function and mitigate disease. The success of this approach depends critically on the methods used to isolate, preserve, and retrieve intact, functional mitochondria. Objective: To optimize an isolation strategy that preserves mitochondrial integrity, dynamics, and metabolic activity and to evaluate conditions that enable short-term storage for future organelle biobanking applications. Methods: We compared a mitochondria isolation method developed in our laboratory (Protocol A) with a commercially available kit (Protocol B). Donor mitochondria were isolated from proximal tubular cells and transplanted into HEK293T recipient cells. Mitochondrial functionality was assessed following transfer into HEK293T cells by measuring reactive oxygen species (MitoSOX Red), oxygen consumption rate (OCR) using Seahorse XF analysis, and high-resolution imaging of mitochondrial morphology and dynamics. We further evaluated mitochondrial storage at low temperature and subsequent functional recovery. Results: Protocol A enabled faster isolation (~30 min) than Protocol B (~80 min) and yielded mitochondria with higher transplantation efficiency, greater OCR, preserved dynamic morphology, and lower oxidative stress. Mitochondria isolated using Protocol A remained metabolically active after transplantation and continued to exhibit fission and fusion, whereas those isolated using Protocol B showed reduced dynamic behavior. Importantly, mitochondria isolated with Protocol A retained functional integrity after low-temperature storage, supporting their potential for standardized preservation. Conclusions: This study presents a robust, efficient, and reproducible isolation and frozen-storage protocol that yields highly functional mitochondria suitable for transplantation. The ability to preserve mitochondrial function after storage further highlights the potential for developing organelle biobanks to support future research and therapeutic applications.