{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T17:51:32Z","timestamp":1789149092725,"version":"build-2803163510"},"reference-count":40,"publisher":"Wiley","issue":"3","license":[{"start":{"date-parts":[[2025,3,19]],"date-time":"2025-03-19T00:00:00Z","timestamp":1742342400000},"content-version":"vor","delay-in-days":18,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["J of Extracellular Vesicle"],"published-print":{"date-parts":[[2025,3]]},"abstract":"<jats:title>ABSTRACT<\/jats:title><jats:p>The isolation of extracellular vesicles (EVs) using currently available methods frequently compromises purity and yield to prioritize speed. Here, we present a next\u2010generation aqueous two\u2010phase system (next\u2010gen ATPS) for the isolation of EVs regardless of scale and volume that is superior to conventional methods such as ultracentrifugation (UC) and commercial kits. This is made possible by the two aqueous phases, one rich in polyethylene glycol (PEG) and the other rich in dextran (DEX), whereby fully encapsulated lipid vesicles preferentially migrate to the DEX\u2010rich phase to achieve a local energy minimum for the EVs. Isolated EVs as found in the DEX\u2010rich phase are more amenable to biomarker analysis such as nanoscale flow cytometry (nFC) when using various pre\u2010conjugated antibodies specific for CD9, CD63 and CD81. TRIzol RNA isolation is further enabled by the addition of dextranase, a critical component of this next\u2010gen ATPS method. RNA yield of next\u2010gen ATPS\u2010isolated EVs is superior to UC and other commercial kits. This negates the use of specialized EV RNA extraction kits. The use of dextranase also enables more accurate immunoreactivity of pre\u2010conjugated antibodies for the detection of EVs by nFC. Transcriptomic analysis of EVs isolated using the next\u2010gen ATPS revealed a strong overlap in microRNA (miRNA), circular RNA (circRNA) and small nucleolar RNA (snoRNA) profiles with EV donor cells, as well as EVs isolated by UC and the exoRNeasy kit, while detecting a superior number of circRNAs compared to the kit in human samples. Overall, this next\u2010gen\u00a0ATPS method stands out as a rapid and highly effective approach to isolate high\u2010quality EVs in high yield, ensuring optimal extraction and analysis of EV\u2010encapsulated nucleic acids.<\/jats:p>","DOI":"10.1002\/jev2.70058","type":"journal-article","created":{"date-parts":[[2025,3,20]],"date-time":"2025-03-20T02:50:45Z","timestamp":1742439045000},"source":"Crossref","is-referenced-by-count":8,"title":["Next Generation Aqueous Two\u2010Phase System for Gentle, Effective, and Timely Extracellular Vesicle Isolation and Transcriptomic Analysis"],"prefix":"10.1002","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-9563-8451","authenticated-orcid":false,"given":"Boyang","family":"Su","sequence":"first","affiliation":[{"name":"Biological Sciences Platform Sunnybrook Research Institute  Toronto Ontario Canada"},{"name":"Temerty Faculty of Medicine, Department of Medical Biophysics University of Toronto  Toronto Ontario Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Morteza","family":"Jeyhani","sequence":"additional","affiliation":[{"name":"Department of Mechanical, Industrial, and Mechatronics Engineering Toronto Metropolitan University  Toronto Ontario Canada"},{"name":"Keenan Research Centre for Biomedical Science St. Michael's Hospital  Toronto Ontario Canada"},{"name":"Institute for Biomedical Engineering, Science and Technology (iBEST)\u2014Toronto Metropolitan University and St. Michael's Hospital  Toronto Ontario Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gobi","family":"Thillainadesan","sequence":"additional","affiliation":[{"name":"Biological Sciences Platform Sunnybrook Research Institute  Toronto Ontario Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Minzhi","family":"Sheng","sequence":"additional","affiliation":[{"name":"Biological Sciences Platform Sunnybrook Research Institute  Toronto Ontario Canada"},{"name":"Temerty Faculty of Medicine, Department of Medical Biophysics University of Toronto  Toronto Ontario Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Reese","family":"Wunsche","sequence":"additional","affiliation":[{"name":"Biological Sciences Platform Sunnybrook Research Institute  Toronto Ontario Canada"},{"name":"Keenan Research Centre for Biomedical Science St. Michael's Hospital  Toronto Ontario Canada"},{"name":"Institute for Biomedical Engineering, Science and Technology (iBEST)\u2014Toronto Metropolitan University and St. Michael's Hospital  Toronto Ontario Canada"},{"name":"Department of Electrical, Computer and Biomedical Engineering Toronto Metropolitan University  Toronto Ontario Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thamara","family":"Dayarathna","sequence":"additional","affiliation":[{"name":"Biological Sciences Platform Sunnybrook Research Institute  Toronto Ontario Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kristin","family":"Cimolai","sequence":"additional","affiliation":[{"name":"Biological Sciences Platform Sunnybrook Research Institute  Toronto Ontario Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hanyi","family":"Weng","sequence":"additional","affiliation":[{"name":"Temerty Faculty of Medicine, Department of Medical Biophysics University of Toronto  Toronto Ontario Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katarzyna J.","family":"Jerzak","sequence":"additional","affiliation":[{"name":"Biological Sciences Platform Sunnybrook Research Institute  Toronto Ontario Canada"},{"name":"Division of Medical Oncology and Hematology, Department of Medicine Odette Cancer Centre Sunnybrook Health Sciences Centre  Toronto Ontario Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stanley K.","family":"Liu","sequence":"additional","affiliation":[{"name":"Biological Sciences Platform Sunnybrook Research Institute  Toronto Ontario Canada"},{"name":"Temerty Faculty of Medicine, Department of Medical Biophysics University of Toronto  Toronto Ontario Canada"},{"name":"Department of Radiation Oncology University of Toronto  Toronto Ontario Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Scott S. H.","family":"Tsai","sequence":"additional","affiliation":[{"name":"Department of Mechanical, Industrial, and Mechatronics Engineering Toronto Metropolitan University  Toronto Ontario Canada"},{"name":"Keenan Research Centre for Biomedical Science St. Michael's Hospital  Toronto Ontario Canada"},{"name":"Institute for Biomedical Engineering, Science and Technology (iBEST)\u2014Toronto Metropolitan University and St. Michael's Hospital  Toronto Ontario Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hon S.","family":"Leong","sequence":"additional","affiliation":[{"name":"Biological Sciences Platform Sunnybrook Research Institute  Toronto Ontario Canada"},{"name":"Temerty Faculty of Medicine, Department of Medical Biophysics University of Toronto  Toronto Ontario Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2025,3,19]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/s0065\u20103233(08)60244\u20102"},{"key":"e_1_2_9_3_1","volume-title":"Partition of Cell Particles and Macromolecules: Separation and Purification of Biomolecules, Cell Organelles, Membranes, and Cells in Aqueous Polymer Two\u2010Phase Systems and Their Use in Biochemical Analysis and Biotechnology","author":"Albertsson P. A.","year":"1986"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.8.3.793"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1385\/MB:20:1:085"},{"issue":"1","key":"e_1_2_9_6_1","first-page":"18","article-title":"Purification of Plasmid and High Molecular Mass DNA Using PEG\u2010Salt Two\u2010Phase Extraction","volume":"11","author":"Cole K.","year":"1991","journal-title":"Biotechniques"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1021\/acsnano.4c00666"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.3402\/jev.v5.32945"},{"key":"e_1_2_9_9_1","doi-asserted-by":"publisher","DOI":"10.1055\/s\u20100038\u20101668544"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF01253770"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.1080\/15422119.2014.983128"},{"key":"e_1_2_9_12_1","doi-asserted-by":"publisher","DOI":"10.1385\/MB:19:3:269"},{"key":"e_1_2_9_13_1","volume-title":"Partitioning in Aqueous Two\u2010Phase Systems: Theory, Methods, Uses, and Applications to Biotechnology","author":"Johansson D.","year":"1985"},{"key":"e_1_2_9_14_1","doi-asserted-by":"publisher","DOI":"10.1016\/0304\u20104165(91)90188\u2010M"},{"key":"e_1_2_9_15_1","doi-asserted-by":"publisher","DOI":"10.1186\/s40486\u2010017\u20100049\u20107"},{"key":"e_1_2_9_16_1","doi-asserted-by":"publisher","DOI":"10.1128\/MMBR.69.2.306\u2010325.2005"},{"key":"e_1_2_9_17_1","doi-asserted-by":"crossref","unstructured":"Kim J. H.Shin J.Kim J.Kim andJ.Park.2015.PLoS ONE10 no.6:e0129460.https:\/\/doi.org\/10.1371\/journal.pone.0129460.","DOI":"10.1371\/journal.pone.0129760"},{"key":"e_1_2_9_18_1","doi-asserted-by":"publisher","DOI":"10.1039\/d2nr01160c"},{"key":"e_1_2_9_19_1","doi-asserted-by":"publisher","DOI":"10.1248\/bpb.b16\u201000891"},{"key":"e_1_2_9_20_1","doi-asserted-by":"publisher","DOI":"10.3402\/jev.v4.29509"},{"key":"e_1_2_9_21_1","doi-asserted-by":"publisher","DOI":"10.1080\/20013078.2017.1359478"},{"key":"e_1_2_9_22_1","doi-asserted-by":"crossref","unstructured":"Mathiazakan P. S. Y.Shing S. S.Ying et\u00a0al.2016.Separation and Purification Technology171:206\u2013213.https:\/\/doi.org\/10.1016\/j.seppur.2016.07.032.","DOI":"10.1016\/j.seppur.2016.07.017"},{"issue":"1","key":"e_1_2_9_23_1","first-page":"43","article-title":"Subfractionationof human Peripheral Blood Lymphocytes by Partitioning in Two\u2010polymer Aqueousphase Systems. Characteristics of a Small Subpopulation of Cells With Highpartition Coefficient","volume":"21","author":"Michalski H.","year":"1986","journal-title":"Journal of Clinical & Laboratory Immunology"},{"key":"e_1_2_9_24_1","doi-asserted-by":"crossref","unstructured":"Nam K. W.Chang andH.Hong.2005.Biomedical Microdevices7:189\u2013195.https:\/\/doi.org\/10.1007\/s10544\u2010005\u20106074\u20102.","DOI":"10.1007\/s10544-005-3025-6"},{"key":"e_1_2_9_25_1","doi-asserted-by":"publisher","DOI":"10.1002\/pros.23764"},{"key":"e_1_2_9_26_1","doi-asserted-by":"publisher","DOI":"10.1016\/0021-9673(94)80105-3"},{"key":"e_1_2_9_27_1","doi-asserted-by":"publisher","DOI":"10.3390\/cells9091955"},{"key":"e_1_2_9_28_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0194818"},{"key":"e_1_2_9_29_1","doi-asserted-by":"publisher","DOI":"10.1038\/nbt0391\u2010252"},{"key":"e_1_2_9_30_1","doi-asserted-by":"publisher","DOI":"10.1007\/s10544\u2010008\u20109238\u20108"},{"key":"e_1_2_9_31_1","doi-asserted-by":"publisher","DOI":"10.1002\/adma.200904271"},{"key":"e_1_2_9_32_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmat2515"},{"key":"e_1_2_9_33_1","doi-asserted-by":"publisher","DOI":"10.1080\/20013078.2018.1535750"},{"key":"e_1_2_9_34_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrm.2017.125"},{"key":"e_1_2_9_35_1","volume-title":"Methods in Enzymology: Aqueous Two\u2010Phase Systems","author":"Walter G.","year":"1994"},{"key":"e_1_2_9_36_1","doi-asserted-by":"publisher","DOI":"10.1016\/0003\u20102697(91)90357\u20102"},{"key":"e_1_2_9_37_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi00659a004"},{"key":"e_1_2_9_38_1","doi-asserted-by":"publisher","DOI":"10.1016\/0005\u20102736(80)90279\u20105"},{"key":"e_1_2_9_39_1","doi-asserted-by":"publisher","DOI":"10.1002\/jev2.12404"},{"key":"e_1_2_9_40_1","doi-asserted-by":"publisher","DOI":"10.1172\/JCI81129"},{"key":"e_1_2_9_41_1","doi-asserted-by":"publisher","DOI":"10.1002\/bit.20276"}],"container-title":["Journal of Extracellular Vesicles"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/isevjournals.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/jev2.70058","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,16]],"date-time":"2025-10-16T13:49:22Z","timestamp":1760622562000},"score":1,"resource":{"primary":{"URL":"https:\/\/isevjournals.onlinelibrary.wiley.com\/doi\/10.1002\/jev2.70058"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,3]]},"references-count":40,"journal-issue":{"issue":"3","published-print":{"date-parts":[[2025,3]]}},"alternative-id":["10.1002\/jev2.70058"],"URL":"https:\/\/doi.org\/10.1002\/jev2.70058","archive":["Portico"],"relation":{},"ISSN":["2001-3078","2001-3078"],"issn-type":[{"value":"2001-3078","type":"print"},{"value":"2001-3078","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,3]]},"article-number":"e70058"}}