{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T11:37:43Z","timestamp":1785411463723,"version":"3.56.0"},"posted":{"date-parts":[[2026,6,17]]},"group-title":"Bioengineering","reference-count":54,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,6,17]],"date-time":"2026-06-17T00:00:00Z","timestamp":1781654400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"award":["7R01NS136752-02"],"award-info":[{"award-number":["7R01NS136752-02"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,6,17]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  A subset of extracellular vehicles (\n                  <jats:bold>EVs<\/jats:bold>\n                  ) with particle diameters &gt;200 nm, large vesicles (\n                  <jats:bold>lEVs<\/jats:bold>\n                  ) contain mitochondria that increase recipient cell bioenergetics. To date, sequential centrifugation (\n                  <jats:bold>SC<\/jats:bold>\n                  ) is the most reported protocol to separate lEVs from the smaller EVs (&lt;200 nm)\/exosomes. We have previously demonstrated that lEVs derived from brain endothelial cells (\n                  <jats:bold>BECs<\/jats:bold>\n                  ) using the standard SC method transferred their innate mitochondria to recipient BECs, increased recipient BEC bioenergetics, reduced brain infarct volume, and improved behavioral outcomes in a mouse model of transient ischemic stroke. Despite their promising therapeutic activity, SC-isolated lEVs are likely a mixture of mitochondria-containing lEVs and non-mitochondria-containing lEVs. We hypothesized that subsequent purification of SC-isolated lEVs using density-gradient centrifugation (\n                  <jats:bold>DGC<\/jats:bold>\n                  ) may yield a purer sample of mitochondria-containing lEVs. We established a DGC protocol to purify lEVs. In this pilot study, lEVs isolated using SC and DGC protocols were compared to determine their physicochemical characteristics and their effects on recipient BEC bioenergetics. SC-lEVs and DGC-lEVs both significantly restored ATP levels in OGD-injured BECs with no difference between groups. However, a Seahorse mitochondrial function assay revealed distinct functional effects: SC-lEVs did not significantly alter respiration, whereas DGC-lEVs induced a dose-dependent increase in oxygen consumption rate, indicating enhanced oxidative phosphorylation. These findings demonstrate that DGC purification yields a more mitochondria-enriched and functionally potent lEV preparation with an enhanced capacity to restore oxidative phosphorylation in ischemic BECs.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Graphical abstract<\/jats:title>\n                  <jats:fig id=\"ufig1\" position=\"float\" orientation=\"portrait\" fig-type=\"figure\">\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"732469v1_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:sec>","DOI":"10.64898\/2026.06.15.732469","type":"posted-content","created":{"date-parts":[[2026,6,17]],"date-time":"2026-06-17T10:00:14Z","timestamp":1781690414000},"source":"Crossref","is-referenced-by-count":0,"title":["Sequential-\n                  <i>vs<\/i>\n                  . density gradient- centrifugation for the isolation of mitochondria-containing extracellular vesicles"],"prefix":"10.64898","author":[{"given":"Kandarp M.","family":"Dave","sequence":"first","affiliation":[{"name":"Graduate School of Pharmaceutical Sciences, Duquesne University, Pittsburgh, PA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bodhi T.","family":"Brady","sequence":"additional","affiliation":[{"name":"Graduate School of Pharmaceutical Sciences, Duquesne University, Pittsburgh, PA"},{"name":"Department of Biology and Microbiology, South Dakota State University, Brookings, SD"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Balaji","family":"Govindaswamy","sequence":"additional","affiliation":[{"name":"Department of Neurology, The University of Texas Health Science Center at Houston, TX"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vivek S.","family":"Basudkar","sequence":"additional","affiliation":[{"name":"Graduate School of Pharmaceutical Sciences, Duquesne University, Pittsburgh, PA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Donna B.","family":"Stolz","sequence":"additional","affiliation":[{"name":"Center for Biologic Imaging, University of Pittsburgh Medical School, Pittsburgh, PA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Devika S","family":"Manickam","sequence":"additional","affiliation":[{"name":"Department of Neurology, The University of Texas Health Science Center at Houston, TX"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026061812500752000_2026.06.15.732469v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.1807"},{"issue":"10","key":"2026061812500752000_2026.06.15.732469v1.2","doi-asserted-by":"crossref","first-page":"3650","DOI":"10.1021\/acs.molpharmaceut.5b00364","article-title":"Exogenous DNA Loading into Extracellular Vesicles via Electroporation is Size-Dependent and Enables Limited Gene Delivery","volume":"12","year":"2015","journal-title":"Molecular pharmaceutics"},{"key":"2026061812500752000_2026.06.15.732469v1.3","doi-asserted-by":"publisher","DOI":"10.1016\/j.biomaterials.2017.07.011"},{"key":"2026061812500752000_2026.06.15.732469v1.4","doi-asserted-by":"publisher","DOI":"10.1038\/s12276-019-0258-7"},{"key":"2026061812500752000_2026.06.15.732469v1.5","doi-asserted-by":"publisher","DOI":"10.3390\/cells8070727"},{"key":"2026061812500752000_2026.06.15.732469v1.6","doi-asserted-by":"crossref","unstructured":"Khan H , Pan J-J , Li Y , Zhang Z , Yang G-Y . Native and Bioengineered Exosomes for Ischemic Stroke Therapy. Frontiers in Cell and Developmental Biology. 2021;9(450).","DOI":"10.3389\/fcell.2021.619565"},{"issue":"7","key":"2026061812500752000_2026.06.15.732469v1.7","doi-asserted-by":"crossref","first-page":"748","DOI":"10.1038\/s41565-021-00931-2","article-title":"Extracellular vesicles as a next-generation drug delivery platform","volume":"16","year":"2021","journal-title":"Nature Nanotechnology"},{"issue":"1","key":"2026061812500752000_2026.06.15.732469v1.8","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1186\/s12951-020-00748-6","article-title":"Synaptosomes: new vesicles for neuronal mitochondrial transplantation","volume":"19","year":"2021","journal-title":"J Nanobiotechnology"},{"key":"2026061812500752000_2026.06.15.732469v1.9","doi-asserted-by":"crossref","first-page":"8472","DOI":"10.1038\/ncomms9472","article-title":"Mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle microRNAs","volume":"6","year":"2015","journal-title":"Nature Communications"},{"key":"2026061812500752000_2026.06.15.732469v1.10","doi-asserted-by":"publisher","DOI":"10.1161\/CIRCRESAHA.118.314601"},{"key":"2026061812500752000_2026.06.15.732469v1.11","doi-asserted-by":"publisher","DOI":"10.1016\/j.jacc.2020.12.060"},{"key":"2026061812500752000_2026.06.15.732469v1.12","doi-asserted-by":"publisher","DOI":"10.1007\/s12195-022-00738-8"},{"key":"2026061812500752000_2026.06.15.732469v1.13","doi-asserted-by":"publisher","DOI":"10.1016\/j.jconrel.2023.01.025"},{"key":"2026061812500752000_2026.06.15.732469v1.14","doi-asserted-by":"crossref","first-page":"803","DOI":"10.1016\/j.jconrel.2024.07.065","article-title":"Mitochondria-containing extracellular vesicles from mouse vs. human brain endothelial cells for ischemic stroke therapy","volume":"373","year":"2024","journal-title":"Journal of Controlled Release"},{"key":"2026061812500752000_2026.06.15.732469v1.15","doi-asserted-by":"publisher","DOI":"10.1016\/j.jconrel.2021.08.038"},{"key":"2026061812500752000_2026.06.15.732469v1.16","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms9472"},{"key":"2026061812500752000_2026.06.15.732469v1.17","doi-asserted-by":"crossref","unstructured":"D\u2019Souza A , Burch A , Dave KM , Sreeram A , Reynolds MJ , Dobbins DX , et al. Microvesicles transfer mitochondria and increase mitochondrial function in brain endothelial cells. Journal of Controlled Release. 2021.","DOI":"10.1101\/2021.04.10.439214"},{"issue":"5","key":"2026061812500752000_2026.06.15.732469v1.18","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1007\/s12195-022-00738-8","article-title":"Engineering Extracellular Vesicles to Modulate Their Innate Mitochondrial Load","volume":"15","year":"2022","journal-title":"Cell Mol Bioeng"},{"key":"2026061812500752000_2026.06.15.732469v1.19","doi-asserted-by":"publisher","DOI":"10.1016\/j.jconrel.2023.01.025"},{"key":"2026061812500752000_2026.06.15.732469v1.20","doi-asserted-by":"publisher","DOI":"10.1183\/13993003.02978-2020"},{"key":"2026061812500752000_2026.06.15.732469v1.21","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pbio.3001166"},{"issue":"1","key":"2026061812500752000_2026.06.15.732469v1.22","doi-asserted-by":"crossref","first-page":"1039","DOI":"10.1038\/s41598-020-57497-7","article-title":"A comparison of methods for the isolation and separation of extracellular vesicles from protein and lipid particles in human serum","volume":"10","year":"2020","journal-title":"Scientific reports"},{"issue":"9","key":"2026061812500752000_2026.06.15.732469v1.23","doi-asserted-by":"crossref","first-page":"e12128","DOI":"10.1002\/jev2.12128","article-title":"Molecular evaluation of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin","volume":"10","year":"2021","journal-title":"Journal of Extracellular Vesicles"},{"issue":"1","key":"2026061812500752000_2026.06.15.732469v1.24","doi-asserted-by":"crossref","first-page":"104","DOI":"10.1186\/s12967-021-02775-9","article-title":"Extracellular vesicles isolated by size-exclusion chromatography present suitability for RNomics analysis in plasma","volume":"19","year":"2021","journal-title":"Journal of Translational Medicine"},{"issue":"1","key":"2026061812500752000_2026.06.15.732469v1.25","doi-asserted-by":"crossref","first-page":"33641","DOI":"10.1038\/srep33641","article-title":"Size-Exclusion Chromatography-based isolation minimally alters Extracellular Vesicles\u2019 characteristics compared to precipitating agents","volume":"6","year":"2016","journal-title":"Scientific Reports"},{"issue":"5","key":"2026061812500752000_2026.06.15.732469v1.26","doi-asserted-by":"crossref","first-page":"1462","DOI":"10.1038\/s41596-023-00811-0","article-title":"Comprehensive isolation of extracellular vesicles and nanoparticles","volume":"18","year":"2023","journal-title":"Nature Protocols"},{"issue":"1","key":"2026061812500752000_2026.06.15.732469v1.27","doi-asserted-by":"crossref","first-page":"33935","DOI":"10.1038\/srep33935","article-title":"A novel affinity-based method for the isolation of highly purified extracellular vesicles","volume":"6","year":"2016","journal-title":"Scientific Reports"},{"key":"2026061812500752000_2026.06.15.732469v1.28","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-4939-7652-2_9"},{"issue":"1","key":"2026061812500752000_2026.06.15.732469v1.29","doi-asserted-by":"crossref","first-page":"1535750","DOI":"10.1080\/20013078.2018.1535750","article-title":"Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines","volume":"7","year":"2018","journal-title":"J Extracell Vesicles"},{"key":"2026061812500752000_2026.06.15.732469v1.30","doi-asserted-by":"crossref","unstructured":"Akbar A , Malekian F , Baghban N , Kodam SP , Ullah M . Methodologies to Isolate and Purify Clinical Grade Extracellular Vesicles for Medical Applications. Cells. 2022;11(2).","DOI":"10.3390\/cells11020186"},{"issue":"3","key":"2026061812500752000_2026.06.15.732469v1.31","doi-asserted-by":"crossref","first-page":"151227","DOI":"10.1016\/j.ejcb.2022.151227","article-title":"Commonly used methods for extracellular vesicles\u2019 enrichment: Implications in downstream analyses and use","volume":"101","year":"2022","journal-title":"European Journal of Cell Biology"},{"key":"2026061812500752000_2026.06.15.732469v1.32","doi-asserted-by":"publisher","DOI":"10.1006\/abio.1994.1350"},{"key":"2026061812500752000_2026.06.15.732469v1.33","doi-asserted-by":"publisher","DOI":"10.1038\/s41556-021-00805-8"},{"key":"2026061812500752000_2026.06.15.732469v1.34","doi-asserted-by":"publisher","DOI":"10.1208\/s12249-020-01892-w"},{"issue":"5","key":"2026061812500752000_2026.06.15.732469v1.35","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1007\/s12195-022-00738-8","article-title":"Engineering Extracellular Vesicles to Modulate Their Innate Mitochondrial Load","volume":"15","year":"2022","journal-title":"Cell Mol Bioeng"},{"key":"2026061812500752000_2026.06.15.732469v1.36","doi-asserted-by":"publisher","DOI":"10.1016\/j.jconrel.2023.01.025"},{"key":"2026061812500752000_2026.06.15.732469v1.37","doi-asserted-by":"crossref","first-page":"54","DOI":"10.1016\/j.redox.2018.06.009","article-title":"Exosomal transfer of mitochondria from airway myeloid-derived regulatory cells to T cells","volume":"18","year":"2018","journal-title":"Redox Biol"},{"key":"2026061812500752000_2026.06.15.732469v1.38","doi-asserted-by":"publisher","DOI":"10.1182\/blood-2013-09-529420"},{"key":"2026061812500752000_2026.06.15.732469v1.39","doi-asserted-by":"crossref","first-page":"803","DOI":"10.1016\/j.jconrel.2024.07.065","article-title":"Mitochondria-containing extracellular vesicles from mouse vs. human brain endothelial cells for ischemic stroke therapy","volume":"373","year":"2024","journal-title":"J Control Release"},{"key":"2026061812500752000_2026.06.15.732469v1.40","doi-asserted-by":"publisher","DOI":"10.1016\/j.jconrel.2021.08.038"},{"key":"2026061812500752000_2026.06.15.732469v1.41","doi-asserted-by":"publisher","DOI":"10.1208\/s12249-020-01892-w"},{"key":"2026061812500752000_2026.06.15.732469v1.42","doi-asserted-by":"publisher","DOI":"10.1208\/s12249-020-01892-w"},{"key":"2026061812500752000_2026.06.15.732469v1.43","doi-asserted-by":"crossref","first-page":"25530","DOI":"10.3402\/jev.v4.25530","article-title":"Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer","volume":"4","year":"2015","journal-title":"J Extracell Vesicles"},{"key":"2026061812500752000_2026.06.15.732469v1.44","doi-asserted-by":"publisher","DOI":"10.1016\/j.jaccao.2021.05.006"},{"key":"2026061812500752000_2026.06.15.732469v1.45","doi-asserted-by":"publisher","DOI":"10.1016\/j.jacc.2020.12.060"},{"key":"2026061812500752000_2026.06.15.732469v1.46","doi-asserted-by":"publisher","DOI":"10.1161\/CIRCRESAHA.118.314601"},{"key":"2026061812500752000_2026.06.15.732469v1.47","doi-asserted-by":"publisher","DOI":"10.1038\/nature18928"},{"key":"2026061812500752000_2026.06.15.732469v1.48","doi-asserted-by":"crossref","first-page":"360","DOI":"10.1016\/j.mex.2015.08.002","article-title":"An accurate, precise method for general labeling of extracellular vesicles","volume":"2","year":"2015","journal-title":"MethodsX"},{"key":"2026061812500752000_2026.06.15.732469v1.49","doi-asserted-by":"publisher","DOI":"10.1161\/STROKEAHA.115.009099"},{"key":"2026061812500752000_2026.06.15.732469v1.50","doi-asserted-by":"publisher","DOI":"10.1038\/s41596-022-00719-1"},{"issue":"2","key":"2026061812500752000_2026.06.15.732469v1.51","doi-asserted-by":"crossref","first-page":"306","DOI":"10.1177\/0271678X251349304","article-title":"Extracellular vesicle-mediated mitochondria delivery: Premise and promise","volume":"46","year":"2026","journal-title":"Journal of Cerebral Blood Flow & Metabolism"},{"issue":"1","key":"2026061812500752000_2026.06.15.732469v1.52","doi-asserted-by":"crossref","first-page":"1736935","DOI":"10.1080\/20013078.2020.1736935","article-title":"Unravelling the proteomic landscape of extracellular vesicles in prostate cancer by density-based fractionation of urine","volume":"9","year":"2020","journal-title":"Journal of Extracellular Vesicles"},{"issue":"1","key":"2026061812500752000_2026.06.15.732469v1.53","doi-asserted-by":"crossref","first-page":"15717","DOI":"10.1038\/s41598-019-52083-y","article-title":"Mitochondria damaged by Oxygen Glucose Deprivation can be Restored through Activation of the PI3K\/Akt Pathway and Inhibition of Calcium Influx by Amlodipine Camsylate","volume":"9","year":"2019","journal-title":"Scientific Reports"},{"issue":"1","key":"2026061812500752000_2026.06.15.732469v1.54","doi-asserted-by":"crossref","first-page":"37959","DOI":"10.1038\/srep37959","article-title":"TDB protects vascular endothelial cells against oxygen-glucose deprivation\/reperfusion-induced injury by targeting miR-34a to increase Bcl-2 expression","volume":"6","year":"2016","journal-title":"Scientific Reports"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.06.15.732469","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,18]],"date-time":"2026-06-18T19:50:17Z","timestamp":1781812217000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.06.15.732469"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,17]]},"references-count":54,"URL":"https:\/\/doi.org\/10.64898\/2026.06.15.732469","relation":{},"subject":[],"published":{"date-parts":[[2026,6,17]]},"subtype":"preprint"}}