| Authors |
Awasthi V, Das A, Bkhache M, Alshambky A, Gerhard GS, Bahmed K, Cashman T,
Bouchareb R
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| Submitted By |
Rihab Bouchareb on 9/9/2026 |
| Status |
Published |
| Journal |
Cells |
| Year |
2026 |
| Date Published |
|
| Volume : Pages |
15 : Not Specified |
| PubMed Reference |
42505388 |
| Abstract |
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.
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