{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,12]],"date-time":"2026-10-12T16:23:14Z","timestamp":1791822194956,"version":"4.3.4"},"reference-count":31,"publisher":"MDPI AG","issue":"14","license":[{"start":{"date-parts":[[2026,7,16]],"date-time":"2026-07-16T00:00:00Z","timestamp":1784160000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Institute of Health","award":["5R01DK135470-03"],"award-info":[{"award-number":["5R01DK135470-03"]}]},{"name":"ISAC-NIH","award":["5U24DK128851-03"],"award-info":[{"award-number":["5U24DK128851-03"]}]},{"name":"American Heart Association Postdoctoral Fellowship"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>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.<\/jats:p>","DOI":"10.3390\/cells15141279","type":"journal-article","created":{"date-parts":[[2026,7,17]],"date-time":"2026-07-17T00:55:59Z","timestamp":1784249759000},"page":"1279","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Optimized Isolation and Cryopreservation of Functional Mitochondria for Transplantation and Therapeutic Applications"],"prefix":"10.3390","volume":"15","author":[{"given":"Vikky","family":"Awasthi","sequence":"first","affiliation":[{"name":"Center for Metabolic Disease Research (CMDR), Department of Cardiovascular Sciences, Lewis Katz School of Medicine (LKSM), Temple University, 3500 North Broad Street, Philadelphia, PA 19140, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aasthika","family":"Das","sequence":"additional","affiliation":[{"name":"Center for Metabolic Disease Research (CMDR), Department of Cardiovascular Sciences, Lewis Katz School of Medicine (LKSM), Temple University, 3500 North Broad Street, Philadelphia, PA 19140, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Meriem","family":"Bkhache","sequence":"additional","affiliation":[{"name":"Center for Metabolic Disease Research (CMDR), Department of Cardiovascular Sciences, Lewis Katz School of Medicine (LKSM), Temple University, 3500 North Broad Street, Philadelphia, PA 19140, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Abeer","family":"Alshambky","sequence":"additional","affiliation":[{"name":"Center for Metabolic Disease Research (CMDR), Department of Cardiovascular Sciences, Lewis Katz School of Medicine (LKSM), Temple University, 3500 North Broad Street, Philadelphia, PA 19140, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Glenn S.","family":"Gerhard","sequence":"additional","affiliation":[{"name":"Department of Medical Genetics and Molecular Biochemistry, Temple University, 3500 North Broad Street, Philadelphia, PA 19140, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Karim","family":"Bahmed","sequence":"additional","affiliation":[{"name":"Center for Inflammation and Lung Research, Department of Cardiovascular Sciences, Lewis Katz School of Medicine at Temple University, Philadelphia, PA 19140, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6238-129X","authenticated-orcid":false,"given":"Timothy","family":"Cashman","sequence":"additional","affiliation":[{"name":"Temple Heart & Vascular Institute, Lewis Katz School of Medicine at Temple University, Philadelphia, PA 19140, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6468-1269","authenticated-orcid":false,"given":"Rihab","family":"Bouchareb","sequence":"additional","affiliation":[{"name":"Center for Metabolic Disease Research (CMDR), Department of Cardiovascular Sciences, Lewis Katz School of Medicine (LKSM), Temple University, 3500 North Broad Street, Philadelphia, PA 19140, USA"},{"name":"Department of Medical Genetics and Molecular Biochemistry, Temple University, 3500 North Broad Street, Philadelphia, PA 19140, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"167803","DOI":"10.1016\/j.bbadis.2025.167803","article-title":"Mitochondria: An overview of their origin, genome, architecture, and dynamics","volume":"1871","author":"Moura","year":"2025","journal-title":"Biochim. Biophys. Acta Mol. Basis Dis."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1038\/s41392-024-01839-8","article-title":"Mitochondrial dysfunction: Mechanisms and advances in therapy","volume":"9","author":"Zong","year":"2024","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1038\/s41467-019-13668-3","article-title":"Mitochondrial TCA cycle metabolites control physiology and disease","volume":"11","author":"Chandel","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"102838","DOI":"10.1016\/j.jbc.2022.102838","article-title":"Regulation and function of the mammalian tricarboxylic acid cycle","volume":"299","author":"Arnold","year":"2023","journal-title":"J. Biol. Chem."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Jimenez-Uribe, A.P., Hernandez-Cruz, E.Y., Ramirez-Magana, K.J., and Pedraza-Chaverri, J. (2021). Involvement of Tricarboxylic Acid Cycle Metabolites in Kidney Diseases. Biomolecules, 11.","DOI":"10.3390\/biom11091259"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"a040592","DOI":"10.1101\/cshperspect.a040592","article-title":"Nucleotide Metabolism","volume":"13","author":"Chandel","year":"2021","journal-title":"Cold Spring Harb. Perspect. Biol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1016\/j.semcancer.2017.06.008","article-title":"The TCA cycle as a bridge between oncometabolism and DNA transactions in cancer","volume":"47","author":"Ciccarone","year":"2017","journal-title":"Semin. Cancer Biol."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Sanchez-Garcia, F.J., Perez-Hernandez, C.A., Rodriguez-Murillo, M., and Moreno-Altamirano, M.M.B. (2021). The Role of Tricarboxylic Acid Cycle Metabolites in Viral Infections. Front. Cell. Infect. Microbiol., 11.","DOI":"10.3389\/fcimb.2021.725043"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"a040584","DOI":"10.1101\/cshperspect.a040584","article-title":"Amino Acid Metabolism","volume":"13","author":"Chandel","year":"2021","journal-title":"Cold Spring Harb. Perspect. Biol."},{"key":"ref_10","first-page":"162","article-title":"Determiners of cell fates: The tricarboxylic acid cycle versus the citrate-malate shuttle","volume":"14","author":"Guo","year":"2023","journal-title":"Protein Cell"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1434","DOI":"10.1038\/s41556-024-01429-4","article-title":"Mitochondria and cell death","volume":"26","author":"Glover","year":"2024","journal-title":"Nat. Cell Biol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"4032","DOI":"10.4161\/cc.10.23.18384","article-title":"Mechanisms of mitochondria and autophagy crosstalk","volume":"10","author":"Rambold","year":"2011","journal-title":"Cell Cycle"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1042\/BJ20081386","article-title":"How mitochondria produce reactive oxygen species","volume":"417","author":"Murphy","year":"2009","journal-title":"Biochem. J."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"3716","DOI":"10.1172\/JCI120849","article-title":"Mitochondrial dysfunction in pathophysiology of heart failure","volume":"128","author":"Zhou","year":"2018","journal-title":"J. Clin. Investig."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"379","DOI":"10.1146\/annurev-physiol-042222-025000","article-title":"Mitochondrial Dysfunction in Kidney Tubulopathies","volume":"86","author":"Hoogstraten","year":"2024","journal-title":"Annu. Rev. Physiol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"e00292","DOI":"10.1016\/j.neurot.2023.10.002","article-title":"Mitochondrial dysfunction in neurodegenerative disorders","volume":"21","author":"Klemmensen","year":"2024","journal-title":"Neurotherapeutics"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"165838","DOI":"10.1016\/j.bbadis.2020.165838","article-title":"Mitochondrial dysfunction in metabolic syndrome","volume":"1866","author":"Prasun","year":"2020","journal-title":"Biochim. Biophys. Acta Mol. Basis Dis."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1281","DOI":"10.1177\/1535370216641787","article-title":"Role of mitochondrial dysfunction in cancer progression","volume":"241","author":"Hsu","year":"2016","journal-title":"Exp. Biol. Med."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"941","DOI":"10.1186\/s12967-024-05752-0","article-title":"Mitochondrial transplantation: A promising strategy for treating degenerative joint diseases","volume":"22","author":"Luo","year":"2024","journal-title":"J. Transl. Med."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"e14","DOI":"10.4110\/in.2022.22.e14","article-title":"Mitochondrial Transplantation Ameliorates the Development and Progression of Osteoarthritis","volume":"22","author":"Lee","year":"2022","journal-title":"Immune Netw."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"lnae017","DOI":"10.1093\/lifemedi\/lnae017","article-title":"Mitochondrial transplantation for the treatment of cardiac and noncardiac diseases: Mechanisms, prospective, and challenges","volume":"3","author":"Wang","year":"2024","journal-title":"Life Med."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1016\/bs.mcb.2019.10.002","article-title":"Isolation of mitochondria from cells and tissues","volume":"155","author":"Liao","year":"2020","journal-title":"Methods Cell Biol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"100243","DOI":"10.1016\/j.xpro.2020.100243","article-title":"Isolation and functional analysis of peridroplet mitochondria from murine brown adipose tissue","volume":"2","author":"Ngo","year":"2021","journal-title":"STAR Protoc."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"3.27.1","DOI":"10.1002\/0471143030.cb0327s68","article-title":"Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane and Detergent Resistant Membrane Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts","volume":"68","author":"Williamson","year":"2015","journal-title":"Curr. Protoc. Cell Biol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"4413","DOI":"10.1113\/jphysiol.2011.212712","article-title":"Mitochondria: Isolation, structure and function","volume":"589","author":"Picard","year":"2011","journal-title":"J. Physiol."},{"key":"ref_26","first-page":"225","article-title":"Assessment of Mitochondrial Fission\/Fusion Dynamics in Kidney Proximal Tubular Cells","volume":"14","author":"Awasthi","year":"2025","journal-title":"J. Vis. Exp."},{"key":"ref_27","first-page":"187","article-title":"Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria","volume":"13","author":"Bouchareb","year":"2022","journal-title":"J. Vis. Exp."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"833","DOI":"10.1002\/dvg.22050","article-title":"Mouse lines with photo-activatable mitochondria to study mitochondrial dynamics","volume":"50","author":"Pham","year":"2012","journal-title":"Genesis"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Franko, A., Baris, O.R., Bergschneider, E., von Toerne, C., Hauck, S.M., Aichler, M., Walch, A.K., Wurst, W., Wiesner, R.J., and Johnston, I.C. (2013). Efficient isolation of pure and functional mitochondria from mouse tissues using automated tissue disruption and enrichment with anti-TOM22 magnetic beads. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0082392"},{"key":"ref_30","first-page":"367","article-title":"Simple, reliable, and time-efficient colorimetric method for the assessment of mitochondrial function and toxicity","volume":"18","author":"Ahmad","year":"2018","journal-title":"Biomol. Biomed."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Jang, S., Chapa-Dubocq, X.R., Fossati, S., and Javadov, S. (2021). Analysis of Mitochondrial Calcium Retention Capacity in Cultured Cells: Permeabilized Cells Versus Isolated Mitochondria. Front. Physiol., 12.","DOI":"10.3389\/fphys.2021.773839"}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/15\/14\/1279\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,24]],"date-time":"2026-07-24T10:50:43Z","timestamp":1784890243000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/15\/14\/1279"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,16]]},"references-count":31,"journal-issue":{"issue":"14","published-online":{"date-parts":[[2026,7]]}},"alternative-id":["cells15141279"],"URL":"https:\/\/doi.org\/10.3390\/cells15141279","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,16]]}}}