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Wharton Jel Kaynaklı Eksozom İzolasyonu: Metot Karşılaştırma Çalışması

Year 2023, Volume: 13 Issue: 1, 152 - 161, 01.03.2023

Dilek Kaan

https://doi.org/10.21597/jist.1200996

Abstract

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.

Keywords

Wharton Jel Eksozom İzolasyon Metotları

Thanks

Bu çalışmanın yapılmasında; laboratuvar olanak ve elverişli çalışma ortamı sunan, Erciyes Üniversitesi, Genom ve Kök Hücre Merkezi’ne teşekkürlerimi sunarım.

References

  • 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

Wharton Gel-Derived Exosome Isolatıon: A Method Comparative Study

Year 2023, Volume: 13 Issue: 1, 152 - 161, 01.03.2023

Dilek Kaan

https://doi.org/10.21597/jist.1200996

Abstract

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

References

  • 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
There are 24 citations in total.

Details

Primary Language Turkish
Subjects Structural Biology
Journal Section Biyoloji / Biology
Authors

Dilek Kaan 0000-0003-3622-2249

Early Pub Date February 24, 2023
Publication Date March 1, 2023
Submission Date November 8, 2022
Acceptance Date December 28, 2022
Published in Issue Year 2023 Volume: 13 Issue: 1

Cite

APA Kaan, D. (2023). Wharton Jel Kaynaklı Eksozom İzolasyonu: Metot Karşılaştırma Çalışması. Journal of the Institute of Science and Technology, 13(1), 152-161. https://doi.org/10.21597/jist.1200996
AMA Kaan D. Wharton Jel Kaynaklı Eksozom İzolasyonu: Metot Karşılaştırma Çalışması. J. Inst. Sci. and Tech. March 2023;13(1):152-161. doi:10.21597/jist.1200996
Chicago Kaan, Dilek. “Wharton Jel Kaynaklı Eksozom İzolasyonu: Metot Karşılaştırma Çalışması”. Journal of the Institute of Science and Technology 13, no. 1 (March 2023): 152-61. https://doi.org/10.21597/jist.1200996.
EndNote Kaan D (March 1, 2023) Wharton Jel Kaynaklı Eksozom İzolasyonu: Metot Karşılaştırma Çalışması. Journal of the Institute of Science and Technology 13 1 152–161.
IEEE D. Kaan, “Wharton Jel Kaynaklı Eksozom İzolasyonu: Metot Karşılaştırma Çalışması”, J. Inst. Sci. and Tech., vol. 13, no. 1, pp. 152–161, 2023, doi: 10.21597/jist.1200996.
ISNAD Kaan, Dilek. “Wharton Jel Kaynaklı Eksozom İzolasyonu: Metot Karşılaştırma Çalışması”. Journal of the Institute of Science and Technology 13/1 (March 2023), 152-161. https://doi.org/10.21597/jist.1200996.
JAMA Kaan D. Wharton Jel Kaynaklı Eksozom İzolasyonu: Metot Karşılaştırma Çalışması. J. Inst. Sci. and Tech. 2023;13:152–161.
MLA Kaan, Dilek. “Wharton Jel Kaynaklı Eksozom İzolasyonu: Metot Karşılaştırma Çalışması”. Journal of the Institute of Science and Technology, vol. 13, no. 1, 2023, pp. 152-61, doi:10.21597/jist.1200996.
Vancouver Kaan D. Wharton Jel Kaynaklı Eksozom İzolasyonu: Metot Karşılaştırma Çalışması. J. Inst. Sci. and Tech. 2023;13(1):152-61.
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