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Cited by 310Extracellular vesicles in diseaseNanoparticle-Based Drug DeliveryCellular transport and secretion

Effects of storage temperature on airway exosome integrity for diagnostic and functional analyses

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Maroto, Rosario, Zhao, Yingxin, Jamaluddin, Mohammad, Popov, Vsevolod L., Wang, Hongwang, Kalubowilage, Madumali, Zhang, Yueqing, Luisi, Jonathan +5 more·2017·Journal of Extracellular Vesicles
DOI 10.1080/20013078.2017.1359478PMC PMC5556670
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Abstract

Background: Extracellular vesicles contain biological molecules specified by cell‐type of origin and modified by microenvironmental changes. To conduct reproducible studies on exosome content and function, storage conditions need to have minimal impact on airway exosome integrity. Aim: We compared surface properties and protein content of airway exosomes that had been freshly isolated vs. those that had been treated with cold storage or freezing. Methods: Mouse bronchoalveolar lavage fluid (BALF) exosomes purified by differential ultracentrifugation were analysed immediately or stored at +4°C or −80°C. Exosomal structure was assessed by dynamic light scattering (DLS), transmission electron microscopy (TEM) and charge density (zeta potential, ζ). Exosomal protein content, including leaking/dissociating proteins, were identified by label‐free LC‐MS/MS. Results: Freshly isolated BALF exosomes exhibited a mean diameter of 95 nm and characteristic morphology. Storage had significant impact on BALF exosome size and content. Compared to fresh, exosomes stored at +4°C had a 10% increase in diameter, redistribution to polydisperse aggregates and reduced ζ. Storage at −80°C produced an even greater effect, resulting in a 25% increase in diameter, significantly reducing the ζ, resulting in multilamellar structure formation. In fresh exosomes, we identified 1140 high‐confidence proteins enriched in 19 genome ontology biological processes. After storage at room temperature, 848 proteins were identified. In preparations stored at +4°C, 224 proteins appeared in the supernatant fraction compared to the wash fractions from freshly prepared exosomes; these proteins represent exosome leakage or dissociation of loosely bound “peri‐exosomal” proteins. In preparations stored at −80°C, 194 proteins appeared in the supernatant fraction, suggesting that distinct protein groups leak from exosomes at different storage temperatures. Conclusions: Storage destabilizes the surface characteristics, morphological features and protein content of BALF exosomes. For preservation of the exosome protein content and representative functional analysis, airway exosomes should be analysed immediately after isolation.

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  • Exosome stability under different pH, temperature and other conditions has been studied and reported.

    “Exosomes stability in different PH, temperature and other conditions has been already studied and reported.”

    From Potential Therapeutic Effects of Exosomes Packed With a miR-21-Sponge Construct in a Rat Model of Glioblastoma (2019) · cites this paper as [62]

    The Introduction and Results report that storage temperature affects airway exosome morphology and protein content. The Materials and methods section says the solution’s pH was not adjusted; this paper does not report testing exosome stability across different pH conditions. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • Exosome modifications under different maintenance conditions and freeze-thawing cycles have been reported.

    “Moreover, exosomes modifications under a different situation of maintenance and with a different cycle of freeze thawing has been already reported (61, 62).”

    From Potential Therapeutic Effects of Exosomes Packed With a miR-21-Sponge Construct in a Rat Model of Glioblastoma (2019) · cites this paper as [62]

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References 51 · Cited by 310

Research works printed but not linked: 49

  1. [1]Thery C, Zitvogel L, Amigorena S. Exosomes: composition, biogenesis and function. Nat Rev Immunol. 2002; 2: 569– 18. 12154376
  2. [2]Colombo M, Raposo G, Théry C Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol. 2014; 30: 255– 289. 25288114
  3. [3]Stremersch S, De Smedt SC, Raemdonck K Therapeutic and diagnostic applications of extracellular vesicles. J Control Release. 2016; 244: 167– 183. 27491882
  4. [4]Busse WW, Lemanske RF Asthma. New England J Med. 2001; 344: 350– 362. 11172168
  5. [5]Akinbami LJ, Moorman JE, Bailey C, et al Trends in asthma prevalence, health care use, and mortality in the USA, 2001-2010. NCHS Data Brief. 2012;94: 1– 8. 22617340
  6. [6]Lambrecht BN, Hammad H The airway epithelium in asthma. Nat Med. 2012; 18: 684– 692. 22561832
  7. [7]Johnston SL Overview of virus-induced airway disease. Proc Am Thorac Soc. 2005; 2: 150– 156. 16113484
  8. [8]Al-Muhsen S, Johnson JR, Hamid Q Remodeling in asthma. J Allergy Clin Immunol. 2011; 128: 451-462; quiz 463-454. 21636119
  9. [9]Tian B, Patrikeev I, Ochoa L, et al NFkappaB mediates mesenchymal transition, remodeling and pulmonary fibrosis in response to chronic inflammation by viral RNA patterns. Am J Respir Cell Mol Biol. 2017;56:506–520. PMC5449514 27911568
  10. [10]Knight DA, Holgate ST The airway epithelium: structural and functional properties in health and disease. Respirology. 2003; 8: 432– 446. 14708552
  11. [11]Whitsett JA, Alenghat T Respiratory epithelial cells orchestrate pulmonary innate immunity. Nat Immunol. 2015; 16: 27– 35. 25521682 PMC4318521
  12. [12]Starkhammar M, Kumlien Georén S, Swedin L, et al Intranasal Administration of poly(I:C) and LPS in BALB/c Mice induces airway hyperresponsiveness and inflammation via different pathways. PLoS ONE. 2012; 7: e32110. 22355412 PMC3280225
  13. [13]Guillot L, Le Goffic R, Bloch S, et al Involvement of toll-like receptor 3 in the immune response of lung epithelial cells to double-stranded RNA and influenza A virus. J Biol Chem. 2005; 280: 5571– 5580. 15579900
  14. [14]Garofalo R, Mei F, Manganaro M, et al Upregulation of class I major histocompatibility complex (MHC) molecules on respiratory syncytial virus (RSV)-infected airway epithelial cells. Am J Respir Crit Care Med. 1994; 149: A987.
  15. [15]Jamaluddin M, Wang S, Garofalo RP, et al IFN-beta mediates coordinate expression of antigen-processing genes in RSV-infected pulmonary epithelial cells. Am J Physiol Lung Cell Mol Physiol. 2001; 280: L248– L257. 11159003
  16. [16]Tian B, Zhao Y, Kalita M, et al CDK9-dependent transcriptional elongation in the innate interferon-stimulated gene response to respiratory syncytial virus infection in airway epithelial cells. J Virol. 2013; 87: 7075– 7092. 23596302 PMC3676079
  17. [17]Zhang Y, Luxon BA, Casola A, et al Expression of respiratory syncytial virus-induced chemokine gene networks in lower airway epithelial cells revealed by cDNA microarrays. J Virol. 2001; 75: 9044– 9058. 11533168 PMC114473
  18. [18]Zhao Y, Jamaluddin M, Zhang Y, et al Systematic analysis of cell-type differences in the epithelial secretome reveals insights into the pathogenesis of respiratory syncytial virus-induced lower respiratory tract infections. J Immunol. 2017; 198: 3345– 3364. 28258195 PMC5380581
  19. [19]Bertolusso R, Tian B, Zhao Y, et al Dynamic cross talk model of the epithelial innate immune response to double-stranded rna stimulation: coordinated dynamics emerging from cell-level noise. PLoS ONE. 2014; 9: e93396. 24710104 PMC3977818
  20. [20]Fang L, Choudhary S, Tian B, et al Ataxia telangiectasia mutated kinase mediates NF-kappaB serine 276 phosphorylation and interferon expression via the IRF7-RIG-I amplification loop in paramyxovirus infection. J Virol. 2015; 89: 2628– 2642. 25520509 PMC4325710
  21. [21]Choudhary S, Boldogh I, Brasier AR Inside-out signaling pathways from nuclear reactive oxygen species control pulmonary innate immunity. J Innate Immun. 2016; 8: 143– 155. 26756522 PMC4801701
  22. [22]Wiktorowicz JE, Jamaluddin M Proteomic analysis of the asthmatic airway. Adv Exp Med Biol. 2014; 795: 221– 232. 24162912
  23. [23]Levanen B, Bhakta NR, Torregrosa Paredes P, et al Altered microRNA profiles in bronchoalveolar lavage fluid exosomes in asthmatic patients. J Allergy Clin Immunol. 2013; 131: 894– 903. 23333113 PMC4013392
  24. [24]Torregrosa Paredes P, Esser J, Admyre C, et al Bronchoalveolar lavage fluid exosomes contribute to cytokine and leukotriene production in allergic asthma. Allergy. 2012; 67: 911– 919. 22620679
  25. [25]Kesimer M, Scull M, Brighton B, et al Characterization of exosome-like vesicles released from human tracheobronchial ciliated epithelium: a possible role in innate defense. FASEB J. 2009; 23: 1858– 1868. 19190083 PMC2698655
  26. [26]Kulshreshtha A, Ahmad T, Agrawal A, et al Proinflammatory role of epithelial cell-derived exosomes in allergic airway inflammation. J Allergy Clin Immunol. 2013; 131: 1194-1203, 1203.e1191-e1114. 23414598
  27. [27]Schageman J, Zeringer E, Li M, et al The complete exosome workflow solution: from isolation to characterization of RNA cargo. Biomed Res Int. 2013; 2013: 253957. 24205503 PMC3800616
  28. [28]Sarker S, Scholz-Romero K, Perez A, et al Placenta-derived exosomes continuously increase in maternal circulation over the first trimester of pregnancy. J Transl Med. 2014; 12: 204. 25104112 PMC4283151
  29. [30]Cvjetkovic A, Lotvall J, Lasser C The influence of rotor type and centrifugation time on the yield and purity of extracellular vesicles. J Extracell Vesicles. 2014; 3 DOI: 10.3402/jev.v3.23111 PMC3967015 24678386 · DOI
  30. [31]Wessel D, Flugge UI A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem. 1984; 138: 141– 143. 6731838
  31. [32]Cox J, Hein MY, Luber CA, et al Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol Cell Proteomics. 2014; 13: 2513– 2526. 24942700 PMC4159666
  32. [33]Cox J, Mann M MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol. 2008; 26: 1367– 1372. 19029910
  33. [34]Tyanova S, Temu T, Sinitcyn P, et al The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat Methods. 2016; 13: 731– 740. 27348712
  34. [35]Gaudet P, Livstone MS, Lewis SE, et al Phylogenetic-based propagation of functional annotations within the Gene Ontology consortium. Brief Bioinform. 2011; 12: 449– 462. 21873635 PMC3178059
  35. [36]Perera AS, Wang H, Basel MT, et al Channel Blocking of MspA Revisited. Langmuir. 2013; 29: 308– 315. 23214433
  36. [38]Raposo G, Stoorvogel W Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol. 2013; 200: 373– 383. 23420871 PMC3575529
  37. [39]Kesimer M, Gupta R Physical characterization and profiling of airway epithelial derived exosomes using light scattering. Methods. 2015; 87: 59– 63. 25823850 PMC4584172
  38. [40]Basel MT, Shrestha TB, Troyer DL, et al Protease-sensitive, polymer-caged liposomes: a method for making highly targeted liposomes using triggered release. ACS Nano. 2011; 5: 2162– 2175. 21314184
  39. [41]Podaru G, Ogden S, Baxter A, et al Pulsed magnetic field induced fast drug release from magneto liposomes via ultrasound generation. J Phys Chem B. 2014; 118: 11715– 11722. 25110807
  40. [42]Fujita Y, Yoshioka Y, Ito S, et al Intercellular communication by extracellular vesicles and their microRNAs in asthma. Clin Ther. 2014; 36: 873– 881. 24909737
  41. [43]Takahashi T, Kubo H The role of microparticles in chronic obstructive pulmonary disease. Int J Chron Obstruct Pulmon Dis. 2014; 9: 303– 314. 24707174 PMC3971913
  42. [44]Lötvall J, Hill AF, Hochberg F, et al Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the international society for extracellular vesicles. J Extracellular Vesicles. 2014;3: DOI: 10.3402/jev.v3.26913. PMC4275645 25536934 · DOI
  43. [45]Wu Y, Deng W, Klinke DJ 2nd Exosomes: improved methods to characterize their morphology, RNA content, and surface protein biomarkers. Analyst. 2015; 140: 6631– 6642. 26332016 PMC4986832
  44. [46]Lee M, Ban -J-J, Im W, et al Influence of storage condition on exosome recovery. Biotechnol Bioprocess Eng. 2016; 21: 299– 304.
  45. [47]Lorincz AM, Timar CI, Marosvari KA, et al Effect of storage on physical and functional properties of extracellular vesicles derived from neutrophilic granulocytes. J Extracell Vesicles. 2014; 3: 25465. 25536933 PMC4275651
  46. [48]Boing AN, Van Der Pol E, Grootemaat AE, et al Single-step isolation of extracellular vesicles by size-exclusion chromatography. J Extracell Vesicles. 2014; 3: DOI: 10.3402/jev.v3.2343. PMC4159761 25279113 · DOI
  47. [49]Dudani JS, Gossett DR, Tse HT, et al Rapid inertial solution exchange for enrichment and flow cytometric detection of microvesicles. Biomicrofluidics. 2015; 9: 014112. 25713694 PMC4320146
  48. [50]Oksvold MP, Neurauter A, Pedersen KW Magnetic bead-based isolation of exosomes. Methods Mol Biol. 2015; 1218: 465– 481. 25319668
  49. [51]Bosch S, De Beaurepaire L, Allard M, et al Trehalose prevents aggregation of exosomes and cryodamage. Sci Rep. 2016; 6: 36162. 27824088 PMC5099918

Authors
Maroto, R., Zhao, Y., Jamaluddin, M., Popov, V. L., Wang, H., Kalubowilage, M., Zhang, Y., Luisi, J., Sun, H., Culbertson, C. T., Bossmann, S. H., Motamedi, M., & Brasier, A. R.
Affiliations
  • The University of Texas Medical Branch at Galveston (US)
  • Kansas State University (US)
Corresponding author
Brasier, Allan R.
ORCID
Venue
Journal of Extracellular Vesicles
Publisher
Wiley
Published
2017-12
Volume
6
Issue
1
Pages
1359478-1359478
ISSN
2001-3078 (ISSN-L)
DOI
10.1080/20013078.2017.1359478
PMC
PMC5556670
OpenAlex
W2748508681
Cited by
310
References
51
Citations by year
2026: 25 · 2025: 44 · 2024: 44 · 2023: 37 · 2022: 44 · 2021: 58 · 2020: 28 · 2019: 14 · 2018: 12 · 2017: 4
FWCI
6.59
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Extracellular Vesicles (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Directorate for Mathematical and Physical Sciences (1361411)
  • Foundation for the National Institutes of Health (UL1TR001439)
  • National Institutes of Health (AI068865)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
  • Physical Sciences › Materials Science › Biomaterials › Nanoparticle-Based Drug Delivery
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
Keywords
storage temperature, bronchoalveolar lavage fluid, zeta potential, cold storage, freezing, extracellular vesicles

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