Dilek KAAN

Wharton Jel Kaynaklı Eksozom İzolasyonu: Metot Karşılaştırma Çalışması

Eksozomlar endozomdan köken almış, immün modülasyonda önemli role sahip, hücrelerarası etkileşimi sağlayan, nano boyutta biyomoleküllerdir. Eksozomlar tetraspanninler, proteinler, Annexin ve Rab proteinleri gibi çok çeşitli zar proteinlerine sahiptirler ve bu içeriklere sahip olmaları ile enfeksiyon, sinir sistemi, kanser ve nörodejeneratif hastalıkların tedavi ve teşhisinde kullanılmaya adaydır. Hemen hemen bütün vücut sıvılarından salınan eksozomların hem homojen hem de çok sayıda izole edilebilmesi teşhis ve tedavi için önemlidir. Bu nedenle eksozomların saflaştırılması ve tanıya yönelik spesifik eksozom izolasyonu tedavi amaçlı üretimi açısından önemli bir basamaktır. Bu çalışmada wharton jel kaynaklı ticari mezenkimal kök hücre kültür besiyerinden (medyum) farklı izolasyon metotları olan ultrasantrifüj, diferansiyel ultrasantrifüj-filtrasyon ve çökeltme (presipitasyon) metodu kullanılarak en çok sayıda ve en saf eksozom izolasyonu yapılması amaçlanmıştır. Eksozomların karakterizasyonu için; görüntülenmesi, saflığı ve boyut analizi her üç metot için belirlenmiştir. Sonuç olarak ultrasantrifüj metodunda, hem diferansiyel ultrasantrifüjfiltrasyon, hem de presipitasyon metoduna göre çok daha fazla sayıda çok daha küçük boyutta eksozom izolasyonu olduğu görülmüştür. Diferansiyel ultrasantrifüj-filtrasyon metodu sonucunda ise eksozom sayısının çok daha az olduğu ancak ultrasantrifüj yöntemi ile elde edilen eksozomlarla aynı saflıkta olduğu görülmüştür. Eksozomların hemen hemen her hastalığın tedavisinde etkin rol almalarından dolayı hastalıkların teşhisinde kullanım kolaylığı sağlamakla birlikte iyi biyo-dağılım, biyouyumluluk ve düşük immünojeniteye sahip olmaları da tedavi aşamasında avantaj sağlayacaktır.

Anahtar Kelimeler:

Wharton Jel, Eksozom, İzolasyon Metotları

Wharton Gel-Derived Exosome Isolatıon: A Method Comparative Study

Exosomes are nano-sized biomolecules that originate from the endosome, have an important role in immune modulation, and provide intercellular interaction. Exosomes have a wide variety of membrane proteins such as tetraspanins, proteins, Annexin and Rab proteins, and they are candidates for use in the treatment and diagnosis of infections, nervous system, cancer and neurodegenerative diseases. It is important for diagnosis and treatment that exosomes released from almost all body fluids can be isolated both homogeneously and in large numbers. Therefore, the purification of exosomes and obtaining specific exosomes for diagnosis is an important step in their production for therapeutic purposes. This study aimed to isolate the most and purest exosomes by using different isolation methods, ultracentrifugation, differential centrifugation-filtration and precipitation from commercial mesenchymal stem cell culture medium derived from wharton gel. Imaging, purity and size analysis of exosome characterization was determined for methods. As a result, it was observed that as a result of the isolation with the ultracentrifuge method, a much larger number of exosomes were isolated compared to both precipitation and differential centrifugationfiltration methods. According to the differential centrifugation-filtration method, it was observed that the number of exosomes was much less, but the exosomes were of the same purity as the ultracentrifuge method. Since exosomes play an active role in treating almost every disease, they provide ease of use in diagnosing diseases, and their good biodistribution, biocompatibility and low immunogenicity will also provide an advantage in the treatment phase.

Keywords:

Wharton Gel, Exosome, Isolation Methods,

PDF

___

Beeravolu, N., McKee, C., Alamri, A., Mikhael, S., Brown, C., Perez-Cruet, M., & Chaudhry, G. R. (2017). Isolation and characterization of mesenchymal stromal cells from human umbilical cord and fetal placenta. Journal of Visualized Experiments, 3(122). https://doi.org/10.3791/55224-v
Bongso, A., & Fong, C.-Y. (2012). The therapeutic potential, challenges and future clinical directions of stem cells from the wharton’s jelly of a the human umbilical cord. Stem Cell Reviews and Reports, 9(2), 226–240. https://doi.org/10.1007/s12015-012-9418-z
Brydson, R., Brown, A., Hodges, C., Abellan, P., & Hondow, N. (2015). Microscopy of nanoparticulate dispersions. Journal of Microscopy, 260(3), 238–247. https://doi.org/10.1111/jmi.12290
Dragovic, R. A., Gardiner, C., Brooks, A. S., Tannetta, D. S., Ferguson, D. J. P., Hole, P., & Sargent, I. L. (2011). Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis. Nanomedicine: Nanotechnology, Biology and Medicine, 7(6), 780–788. https://doi.org/10.1016/j.nano.2011.04.003
Gangoda, L., Boukouris, S., Liem, M., Kalra, H., & Mathivanan, S. (2014). Extracellular vesicles including exosomes are mediators of signal transduction: Are they protective or pathogenic? Proteomics, 15(2–3), 260–271. https://doi.org/10.1002/pmic.201400234
Greening, D. W., Gopal, S. K., Xu, R., Simpson, R. J., & Chen, W. (2015). Exosomes and their roles in immune regulation and cancer. Seminars in Cell & Developmental Biology, 40, 72–81. https://doi.org/10.1016/j.semcdb.2015.02.009 György, B., Szabó, T. G., Pásztói, M., Pál, Z., Misják, P., Aradi, B., & Buzas, E. I. (2011). Membrane vesicles, current state-of-the-art: Emerging role of extracellular vesicles. Cellular and Molecular Life Sciences, 68(16), 2667–2688. https://doi.org/10.1007/s00018-011-0689-3
Harding, C., Heuser, J., & Stahl, P. (1983). Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes. Journal of Cell Biology, 97(2), 329–339. https://doi.org/10.1083/jcb.97.2.329
Hassan, G., Kasem, I., Soukkarieh, C., & Aljamali, M. (2017). A simple method to isolate and expand human umbilical cord derived mesenchymal stem cells: Using explant method and umbilical cord blood serum. International Journal of Stem Cells, 10(2), 184–192. https://doi.org/10.15283/ijsc17028
Johnstone, R. M., Adam, M., Hammond, J. R., Orr, L., & Turbide, C. (1987). Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes). Journal of Biological Chemistry, 262(19), 9412–9420. https://doi.org/10.1016/s0021-9258(18)48095-7
Karaöz, E., Çetinalp Demircan, P., Erman, G., Güngörürler, E., & Eker Sarıboyacı, A. (2016). Comparative Analyses of Immune -Suppressive Characteristics of Bone-Marrow, Wharton’s Jelly a nd Adipose-Tissue Derived Human MSCs. Turkish Journal of Hematology, 34(3). https://doi.org/10.4274/tjh.2016.0171
Mittelbrunn, M., Gutiérrez-Vázquez, C., Villarroya-Beltri, C., González, S., Sánchez-Cabo, F., González, M. Á., … Sánchez-Madrid, F. (2011). Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells. Nature Communications, 2(1). https://doi.org/10.1038/ncomms1285
Murk, J.L., Stoorvogel, W., Kleijmeer, M. J., & Geuze, H. J. (2002). The plasticity of multivesicular bodies and the regulation of antigen presentation. Seminars in Cell & Developmental Biology, 13(4), 303–311. https://doi.org/10.1016/s1084952102000605
Pirjali, T., Azarpira, N., Ayatollahi, M., Aghdaie, M.H., Geramizadeh, B., & Talai, T., (2013). Isolation and characterization of human mesenchymal stem cells derived from human umbilical cord Wharton’s jelly and amniotic membrane Int. J. Org. Transplant. Med. 4 (3) 111 – 116.
Rasmussen, M. K., Pedersen, J. N., & Marie, R. (2020). Size and surface charge characterization of nanoparticles with a salt gradient. Nature Communications, 11(1). https://doi.org/10.1038/s41467-020-15889-3
Simpson, R. J., Lim, J. W., Moritz, R. L., & Mathivanan, S. (2009). Exosomes: Proteomic insights and diagnostic potential. Expert Review of Proteomics, 6(3), 267–283. https://doi.org/10.1586/epr.09.17
Sun, D., Zhuang, X., Grizzle, W., Miller, D., & Zhang, H.-G. (2011). Abstract 4446: A novel nanoparticle drug delivery system: The anti-inflammatory activity of curcumin is enhanced when encapsulated in exosomes. Cancer Research, 71(8_Supplement), 4446–4446. https://doi.org/10.1158/1538-7445.am2011-4446
Thery, C., Amigorena, S., Raposo, G., & Clayton, A. (2006). Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Current Protocols in Cell Biology, 30(1). https://doi.org/10.1002/0471143030.cb0322s30
Thery, C., Ostrowski, M., & Segura, E. (2009). Membrane vesicles as conveyors of immune responses. Nature Reviews Immunology, 9(8), 581–593. https://doi.org/10.1038/nri2567
Tofino-Vian, M., Guillén, M. I., Pérez del Caz, M. D., Castejón, M. A., & Alcaraz, M. J. (2017). Extracellular vesicles from adipose-derived mesenchymal stem cells downregulate senescence features in osteoarthritic osteoblasts. Oxidative Medicine and Cellular Longevity, 2017, 1–12. https://doi.org/10.1155/2017/7197598 Troyer, D. L., & Weiss, M. L. (2007). Concise review: Wharton’s jelly-derived cells are a primitive stromal cell population. Stem Cells, 26(3), 591–599. https://doi.org/10.1634/stemcells.2007-0439
Vergauwen, G., Dhondt, B., Van Deun, J., De Smedt, E., Berx, G., Timmerman, E., & Hendrix, A. (2017). Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research. Scientific Reports, 7(1). https://doi.org/10.1038/s41598-017-02599-y
Vidal, M., Sainte-Marie, J., Philippot, J. R., & Bienvenue, A. (1989). Asymmetric distribution of phospholipids in the membrane of vesicles released during in vitro maturation of guinea pig reticulocytes: Evidence precluding a role for? aminophospholipid translocase? Journal of Cellular Physiology, 140(3), 455–462. https://doi.org/10.1002/jcp.1041400308
Widowati, W., Wijaya, L., Bachtiar, I., Gunanegara, R. F., Sugeng, S. U., Irawan, Y. A., & Widodo, A.M. (2014). Effect of oxygen tension on proliferation and characteristics of Wharton’s jelly-derived mesenchymal stem cells. Biomarkers and Genomic Medicine, 6(1), 43–48. https://doi.org/10.1016/j.bgm.2014.02.001
Yusoff, Z., Maqbool, M., George, E., Hassan, R.M.D & Ramasamy, R. (2016). Generation and characterisation of human umbilical cord derived mesenchymal stem cells by explant method Medical Journal of Malaysia, 71 (3) 105-110.
Zhang, Y., Liu, Y., Liu, H., & Tang, W. H. (2019). Exosomes: Biogenesis, biologic function and clinical potential. Cell & Bioscience, 9(1). https://doi.org/10.1186/s13578-019-0282-2