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Extracellular Vesicle Isolation and Characterization for Applications in Cartilage Tissue Engineering and Osteoarthritis Therapy

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Cartilage Tissue Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2598))

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Abstract

Extracellular vesicles (EVs) have the capacity for use in cartilage tissue engineering by stimulating tissue repair and microenvironmental reprogramming. This makes them ideal candidates for treating focal cartilage defects and cartilage degeneration in osteoarthritis (OA). Observational studies have reported beneficial biological effects of EVs, such as inhibition of inflammation, enhanced extracellular matrix deposition, and reduced cartilage degradation. Isolation of EVs derived from different source materials such as conditioned cell culture media or biofluids is essential to attribute observed biological effects to EVs as genuine effectors. This chapter presents a density- and a size-based method as well as a combination of both for isolation of EVs from conditioned cell culture media or biofluids. In addition, three methods for characterization of isolated EVs are suggested based on physical properties, protein profiling, and ultrastructural morphology.

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References

  1. Heldring N, Mäger I, Wood MJ et al (2015) Therapeutic potential of multipotent mesenchymal stromal cells and their extracellular vesicles. Hum Gene Ther 26(8):506–517. https://doi.org/10.1089/hum.2015.072

    Article  CAS  PubMed  Google Scholar 

  2. Mancuso P, Raman S, Glynn A et al (2019) Mesenchymal stem cell therapy for osteoarthritis: the critical role of the cell secretome. Front Bioeng Biotechnol 7:9–9. https://doi.org/10.3389/fbioe.2019.00009

    Article  PubMed  PubMed Central  Google Scholar 

  3. Kong L, Zheng L-Z, Qin L et al (2017) Role of mesenchymal stem cells in osteoarthritis treatment. J Orthop Transl 9:89–103. https://doi.org/10.1016/j.jot.2017.03.006

    Article  Google Scholar 

  4. Song Y, Zhang J, Xu H et al (2020) Mesenchymal stem cells in knee osteoarthritis treatment: a systematic review and meta-analysis. J Orthop Transl 24:121–130. https://doi.org/10.1016/j.jot.2020.03.015

    Article  Google Scholar 

  5. O’Shea C, Hynes SO, Shaw G et al (2008) 271. Adverse effects of mesenchymal stem cell transplantation in a denuded Rabbit Carotid artery. Mol Ther 16:S102. https://doi.org/10.1016/S1525-0016(16)39674-5

    Article  Google Scholar 

  6. Wyles CC, Houdek MT, Behfar A et al (2015) Mesenchymal stem cell therapy for osteoarthritis: current perspectives. Stem Cells Clon Adv Appl 8:117–124. https://doi.org/10.2147/sccaa.s68073

    Article  Google Scholar 

  7. Lukomska B, Stanaszek L, Zuba-Surma E et al (2019) Challenges and controversies in human mesenchymal stem cell therapy. Stem Cells Int 2019:9628536. https://doi.org/10.1155/2019/9628536

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Musiał-Wysocka A, Kot M, Majka M (2019) The pros and cons of mesenchymal stem cell-based therapies. Cell Transplant 28(7):801–812. https://doi.org/10.1177/0963689719837897

    Article  PubMed  PubMed Central  Google Scholar 

  9. Bruno S, Deregibus MC, Camussi G (2015) The secretome of mesenchymal stromal cells: role of extracellular vesicles in immunomodulation. Immunol Lett 168(2):154–158. https://doi.org/10.1016/j.imlet.2015.06.007

    Article  CAS  PubMed  Google Scholar 

  10. Bian S, Zhang L, Duan L et al (2014) Extracellular vesicles derived from human bone marrow mesenchymal stem cells promote angiogenesis in a rat myocardial infarction model. J Mol Med (Berl) 92(4):387–397. https://doi.org/10.1007/s00109-013-1110-5

    Article  CAS  Google Scholar 

  11. ter Huurne M, Schelbergen R, Blattes R et al (2012) Antiinflammatory and chondroprotective effects of intraarticular injection of adipose-derived stem cells in experimental osteoarthritis. Arthritis Rheum 64(11):3604–3613. https://doi.org/10.1002/art.34626

    Article  CAS  PubMed  Google Scholar 

  12. van der Pol E, Böing AN, Harrison P et al (2012) Classification, functions, and clinical relevance of extracellular vesicles. Pharmacol Rev 64(3):676–705. https://doi.org/10.1124/pr.112.005983

    Article  CAS  PubMed  Google Scholar 

  13. Rubin O, Canellini G, Delobel J et al (2012) Red blood cell microparticles: clinical relevance. Transfus Med Hemother 39(5):342–347. https://doi.org/10.1159/000342228

    Article  PubMed  PubMed Central  Google Scholar 

  14. De Jong OG, Van Balkom BWM, Schiffelers RM et al (2014) Extracellular vesicles: potential roles in regenerative medicine. Front Immunol 5(608). https://doi.org/10.3389/fimmu.2014.00608

  15. Yáñez-Mó M, Siljander PRM, Andreu Z et al (2015) Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicl 4:27066–27066. https://doi.org/10.3402/jev.v4.27066

    Article  Google Scholar 

  16. Thery C, Witwer KW, Aikawa E et al (2018) 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. J Extracell Vesicl 7(1):1535750. https://doi.org/10.1080/20013078.2018.1535750

    Article  Google Scholar 

  17. Wyciszkiewicz A, Kalinowska-Łyszczarz A, Nowakowski B et al (2019) Expression of small heat shock proteins in exosomes from patients with gynecologic cancers. Sci Rep 9(1):9817. https://doi.org/10.1038/s41598-019-46221-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Chanteloup G, Cordonnier M, Isambert N et al (2020) Membrane-bound exosomal HSP70 as a biomarker for detection and monitoring of malignant solid tumours: a pilot study. Pilot Feasibil Stud 6(1):35. https://doi.org/10.1186/s40814-020-00577-2

    Article  Google Scholar 

  19. Andreu Z, Yáñez-Mó M (2014) Tetraspanins in extracellular vesicle formation and function. Front Immunol 5:442–442. https://doi.org/10.3389/fimmu.2014.00442

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Iavello A, Frech VS, Gai C et al (2016) Role of Alix in miRNA packaging during extracellular vesicle biogenesis. Int J Mol Med 37(4):958–966. https://doi.org/10.3892/ijmm.2016.2488

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Raposo G, Stoorvogel W (2013) Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol 200(4):373

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Tissot J-D, Canellini G, Rubin O et al (2013) Blood microvesicles: from proteomics to physiology. Transl Proteom 1(1):38–52. https://doi.org/10.1016/j.trprot.2013.04.004

    Article  CAS  Google Scholar 

  23. Wolf P (1967) The nature and significance of platelet products in human plasma. Br J Haematol 13(3):269–288. https://doi.org/10.1111/j.1365-2141.1967.tb08741.x

    Article  CAS  PubMed  Google Scholar 

  24. O’Brien JR (1955) The platelet-like activity of serum. Br J Haematol 1(2):223–228

    Article  PubMed  Google Scholar 

  25. Harding C, Stahl P (1983) Transferrin recycling in reticulocytes: pH and iron are important determinants of ligand binding and processing. Biochem Biophys Res Commun 113(2):650–658. https://doi.org/10.1016/0006-291x(83)91776-x

    Article  CAS  PubMed  Google Scholar 

  26. Pan BT, Johnstone RM (1983) Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor. Cell 33(3):967–978. https://doi.org/10.1016/0092-8674(83)90040-5

    Article  CAS  PubMed  Google Scholar 

  27. Anderson HC (1969) Vesicles associated with calcification in the matrix of epiphyseal cartilage. J Cell Biol 41(1):59–72. https://doi.org/10.1083/jcb.41.1.59

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Derfus BA, Kurtin SM, Camacho NP et al (1996) Comparison of matrix vesicles derived from normal and osteoarthritic human articular cartilage. Connect Tissue Res 35(1-4):337–342. https://doi.org/10.3109/03008209609029209

    Article  CAS  PubMed  Google Scholar 

  29. Rosenthal AK, Gohr CM, Ninomiya J et al (2011) Proteomic analysis of articular cartilage vesicles from normal and osteoarthritic cartilage. Arthritis Rheum 63(2):401–411. https://doi.org/10.1002/art.30120

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Iwai K, Minamisawa T, Suga K et al (2016) Isolation of human salivary extracellular vesicles by iodixanol density gradient ultracentrifugation and their characterizations. J Extracell Vesicl 5(1):30829. https://doi.org/10.3402/jev.v5.30829

    Article  CAS  Google Scholar 

  31. Merchant ML, Rood IM, Deegens JKJ et al (2017) Isolation and characterization of urinary extracellular vesicles: implications for biomarker discovery. Nat Rev Nephrol 13(12):731–749. https://doi.org/10.1038/nrneph.2017.148

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. van Herwijnen MJC, Zonneveld MI, Goerdayal S et al (2016) Comprehensive proteomic analysis of human milk-derived extracellular vesicles unveils a novel functional proteome distinct from other milk components*. Mol Cell Proteomics 15(11):3412–3423. https://doi.org/10.1074/mcp.M116.060426

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Foers AD, Chatfield S, Dagley LF et al (2018) Enrichment of extracellular vesicles from human synovial fluid using size exclusion chromatography. J Extracell Vesicl 7(1):1490145. https://doi.org/10.1080/20013078.2018.1490145

    Article  CAS  Google Scholar 

  34. Serrano-Pertierra E, Oliveira-Rodríguez M, Rivas M et al (2019) Characterization of plasma-derived extracellular vesicles isolated by different methods: a comparison study. Bioengineering (Basel) 6(1). https://doi.org/10.3390/bioengineering6010008

  35. Onódi Z, Pelyhe C, Terézia Nagy C et al (2018) Isolation of high-purity extracellular vesicles by the combination of iodixanol density gradient ultracentrifugation and Bind-Elute chromatography from blood plasmA. Front Physiol 9:1479��1479. https://doi.org/10.3389/fphys.2018.01479

    Article  PubMed  PubMed Central  Google Scholar 

  36. Johnstone RM, Mathew A, Mason AB et al (1991) Exosome formation during maturation of mammalian and avian reticulocytes: evidence that exosome release is a major route for externalization of obsolete membrane proteins. J Cell Physiol 147(1):27–36. https://doi.org/10.1002/jcp.1041470105

    Article  CAS  PubMed  Google Scholar 

  37. Miyaki S, Lotz MK (2018) Extracellular vesicles in cartilage homeostasis and osteoarthritis. Curr Opin Rheumatol 30(1):129–135. https://doi.org/10.1097/BOR.0000000000000454

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Todorova D, Simoncini S, Lacroix R et al (2017) Extracellular vesicles in angiogenesis. Circ Res 120(10):1658–1673. https://doi.org/10.1161/circresaha.117.309681

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Becker A, Thakur BK, Weiss JM et al (2016) Extracellular vesicles in cancer: cell-to-cell mediators of metastasis. Cancer Cell 30(6):836–848. https://doi.org/10.1016/j.ccell.2016.10.009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Silva AM, Teixeira JH, Almeida MI et al (2017) Extracellular vesicles: immunomodulatory messengers in the context of tissue repair/regeneration. Eur J Pharm Sci 98:86–95. https://doi.org/10.1016/j.ejps.2016.09.017

    Article  CAS  PubMed  Google Scholar 

  41. Kato T, Miyaki S, Ishitobi H et al (2014) Exosomes from IL-1beta stimulated synovial fibroblasts induce osteoarthritic changes in articular chondrocytes. Arthritis Res Ther 16(4):R163. https://doi.org/10.1186/ar4679

    Article  PubMed  PubMed Central  Google Scholar 

  42. Vonk LA, van Dooremalen SFJ, Liv N et al (2018) Mesenchymal stromal/stem cell-derived extracellular vesicles promote human Cartilage regeneration in vitro. Theranostics 8(4):906–920. https://doi.org/10.7150/thno.20746

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Kim M, Steinberg DR, Burdick JA et al (2019) Extracellular vesicles mediate improved functional outcomes in engineered cartilage produced from MSC/chondrocyte cocultures. Proc Natl Acad Sci U S A 116(5):1569–1578. https://doi.org/10.1073/pnas.1815447116

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Cosenza S, Ruiz M, Toupet K et al (2017) Mesenchymal stem cells derived exosomes and microparticles protect cartilage and bone from degradation in osteoarthritis. Sci Rep 7(1):16214. https://doi.org/10.1038/s41598-017-15376-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Tofino-Vian M, Guillen MI, Perez Del Caz MD et al (2018) Microvesicles from human Adipose tissue-derived mesenchymal stem cells as a new protective strategy in osteoarthritic chondrocytes. Cell Physiol Biochem 47(1):11–25. https://doi.org/10.1159/000489739

    Article  CAS  PubMed  Google Scholar 

  46. Wang Y, Yu D, Liu Z et al (2017) Exosomes from embryonic mesenchymal stem cells alleviate osteoarthritis through balancing synthesis and degradation of cartilage extracellular matrix. Stem Cell Res Ther 8(1):189. https://doi.org/10.1186/s13287-017-0632-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Xiang C, Yang K, Liang Z et al (2018) Sphingosine-1-phosphate mediates the therapeutic effects of bone marrow mesenchymal stem cell-derived microvesicles on articular cartilage defect. Transl Res 193:42–53. https://doi.org/10.1016/j.trsl.2017.12.003

    Article  CAS  PubMed  Google Scholar 

  48. Filardo G, Kon E, Roffi A et al (2015) Platelet-rich plasma: why intra-articular? A systematic review of preclinical studies and clinical evidence on PRP for joint degeneration. Knee Surg Sports Traumatol Arthrosc 23(9):2459–2474. https://doi.org/10.1007/s00167-013-2743-1

    Article  CAS  PubMed  Google Scholar 

  49. Filardo G, Previtali D, Napoli F et al (2020) PRP injections for the treatment of knee osteoarthritis: a meta-analysis of randomized controlled trials. Cartilage 194760352093117. https://doi.org/10.1177/1947603520931170

  50. Kon E, Mandelbaum BR, Buda RE et al (2011) Platelet-rich plasma intra-articular injection versus hyaluronic acid viscosupplementation as treatments for cartilage pathology: from early degeneration to osteoarthritis. Arthroscopy J Arthroscop Relat Surg 27(11):1490–1501

    Article  Google Scholar 

  51. Sillat T, Barreto G, Clarijs P et al (2013) Toll-like receptors in human chondrocytes and osteoarthritic cartilage. Acta Orthop 84(6):585–592. https://doi.org/10.3109/17453674.2013.854666

    Article  PubMed  PubMed Central  Google Scholar 

  52. Miller YI (2005) Toll-like receptors and atherosclerosis: oxidized LDL as an endogenous Toll-like receptor ligand. Future Cardiol 1(6):785–792. https://doi.org/10.2217/14796678.1.6.785

    Article  CAS  PubMed  Google Scholar 

  53. Bobacz K, Sunk IG, Hofstaetter JG et al (2007) Toll-like receptors and chondrocytes: the lipopolysaccharide-induced decrease in cartilage matrix synthesis is dependent on the presence of toll-like receptor 4 and antagonized by bone morphogenetic protein 7. Arthritis Rheum 56(6):1880–1893. https://doi.org/10.1002/art.22637

    Article  CAS  PubMed  Google Scholar 

  54. O’Donnell C, Migliore E, Grandi FC et al (2019) Platelet-Rich Plasma (PRP) from older males with knee osteoarthritis depresses chondrocyte metabolism and upregulates inflammation. J Orthop Res 37(8):1760–1770. https://doi.org/10.1002/jor.24322

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. O’Shaughnessey K, Matuska A, Hoeppner J et al (2014) Autologous protein solution prepared from the blood of osteoarthritic patients contains an enhanced profile of anti-inflammatory cytokines and anabolic growth factors. J Orthop Res 32(10):1349–1355. https://doi.org/10.1002/jor.22671

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Vickers KC, Palmisano BT, Shoucri BM et al (2011) MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol 13(4):423–433. https://doi.org/10.1038/ncb2210

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Vickers KC, Remaley AT (2012) Lipid-based carriers of microRNAs and intercellular communication. Curr Opin Lipidol 23(2):91–97. https://doi.org/10.1097/MOL.0b013e328350a425

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Thery C, Amigorena S, Raposo G et al (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol Chapter 3:Unit 3.22. https://doi.org/10.1002/0471143030.cb0322s30

    Article  PubMed  Google Scholar 

  59. Pourakbari R, Khodadadi M, Aghebati-Maleki A et al (2019) The potential of exosomes in the therapy of the cartilage and bone complications; emphasis on osteoarthritis. Life Sci 236:116861. https://doi.org/10.1016/j.lfs.2019.116861

    Article  CAS  PubMed  Google Scholar 

  60. Otahal A, Kramer K, Kuten-Pella O et al (2020) Characterization and chondroprotective effects of extracellular vesicles from plasma- and serum-based autologous blood-derived products for osteoarthritis therapy. Front Bioeng Biotechnol 8(1114). https://doi.org/10.3389/fbioe.2020.584050

  61. Momen-Heravi F, Balaj L, Alian S et al (2012) Impact of biofluid viscosity on size and sedimentation efficiency of the isolated microvesicles. Front Physiol 3:162. https://doi.org/10.3389/fphys.2012.00162

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Linares R, Tan S, Gounou C et al (2015) High-speed centrifugation induces aggregation of extracellular vesicles. J Extracell Vesicl 4(1):29509. https://doi.org/10.3402/jev.v4.29509

    Article  CAS  Google Scholar 

  63. Monguió-Tortajada M, Gálvez-Montón C, Bayes-Genis A et al (2019) Extracellular vesicle isolation methods: rising impact of size-exclusion chromatography. Cell Mol Life Sci 76(12):2369–2382. https://doi.org/10.1007/s00018-019-03071-y

    Article  CAS  PubMed  Google Scholar 

  64. Zonneveld MI, Brisson AR, van Herwijnen MJC et al (2014) Recovery of extracellular vesicles from human breast milk is influenced by sample collection and vesicle isolation procedures. J Extracell Vesicl 3(1):24215. https://doi.org/10.3402/jev.v3.24215

    Article  CAS  Google Scholar 

  65. Poon C (2020) Measuring the density and viscosity of culture media for optimized computational fluid dynamics analysis of in vitro devices. bioRxiv:2020.2008.2025.266221. https://doi.org/10.1101/2020.08.25.266221

  66. Otahal A, Kuten-Pella O, Kramer K et al (2021) Functional repertoire of EV-associated miRNA profiles after lipoprotein depletion via ultracentrifugation and size exclusion chromatography from autologous blood products. Sci Rep 11(1):5823. https://doi.org/10.1038/s41598-021-84234-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Boere J, van de Lest CH, Libregts SF et al (2016) Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles. J Extracell Vesicles 5:31751. https://doi.org/10.3402/jev.v5.31751

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Center for Regenerative Medicine, University For Continuing Education, Krems, Austria

    Alexander Otahal, Andrea De Luna & Stefan Nehrer

  2. Department of Regenerative Medicine, State Research Institute Centre for Innovative Medicine, Vilnius, Lithuania

    Ali Mobasheri

  3. Research Unit of Medical Imaging, Physics and Technology, Faculty of Medicine, University of Oulu, Oulu, Finland

    Ali Mobasheri

  4. Departments of Orthopedics, Rheumatology and Clinical Immunology, University Medical Center Utrecht, Utrecht, The Netherlands

    Ali Mobasheri

  5. Department of Joint Surgery, First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, China

    Ali Mobasheri

  6. World Health Organization Collaborating Center for Public Health Aspects of Musculoskeletal Health and Aging, Université de Liège, Liège, Belgium

    Ali Mobasheri

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  1. Alexander Otahal
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  2. Andrea De Luna
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  3. Ali Mobasheri
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  4. Stefan Nehrer
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Corresponding authors

Correspondence to Ali Mobasheri or Stefan Nehrer .

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Editors and Affiliations

  1. AO Research Institute Davos, Davos Platz, Switzerland

    Martin J. Stoddart

  2. AO Research Institute Davos, Davos Platz, Switzerland

    Elena Della Bella

  3. UCB Pharma, Slough, UK

    Angela R. Armiento

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Otahal, A., De Luna, A., Mobasheri, A., Nehrer, S. (2023). Extracellular Vesicle Isolation and Characterization for Applications in Cartilage Tissue Engineering and Osteoarthritis Therapy. In: Stoddart, M.J., Della Bella, E., Armiento, A.R. (eds) Cartilage Tissue Engineering. Methods in Molecular Biology, vol 2598. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2839-3_10

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  • DOI: https://doi.org/10.1007/978-1-0716-2839-3_10

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