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BMC veterinary research2019; 15(1); 42; doi: 10.1186/s12917-019-1789-9

Exosomes isolation and identification from equine mesenchymal stem cells.

Abstract: Mesenchymal stem cells are used for different therapeutic approaches, e.g. for osteoarthritis, lesions of the tendon as well as for bone defects. Current research on the mechanism of stem cells on the repair of damaged tissue suggest an important role of a cell-to-cell communication through secreted extracellular vesicles, mainly represented by exosomes. To enhance the scarce knowledge on the functional role of exosomes we compared as a first step different techniques to isolate and identify exosomes from the supernatant of equine adipose derived mesenchymal stem cells for further characterization and usage in functional assays. Results: It was possible to obtain exosomes secreted from equine adipose derived mesenchymal stem cells with three common techniques: a stepwise ultracentrifugation at 100.000 g, an ultrafiltration with 3 kDa exclusion membranes and a charge-based precipitation method. The mean sizes and amounts of exosomes isolated with the different techniques were measured by the nanoparticle tracking analysis. The diameter ranged between 116.2 nm (ultracentrifugation), 453.1 nm (precipitation) and 178.7 nm (ultrafiltration), the counts of particles / ml ranged between 9.6 × 10 (ultracentrifugation), 2.02 × 10 (precipitation) and 52.5 × 10 (ultrafiltration). Relevant marker for exosomes, tetraspanins CD9, CD63 and CD81 were detectable by immunofluorescence staining of the investigated exosomes secreting mesenchymal stem cells. In addition, transmission electron microscopy and immunogold labeling with CD9 and CD90 was performed to display the morphological shape of exosomes and existence of marker relevant for exosomes (CD9) and mesenchymal stem cells (CD90). Western blot analysis of CD9 and CD90 of exosomes ensured the specificity of the rare available respectively cross reacting antibodies against equine antigens. Conclusions: Exosomes generated by equine mesenchymal stem cells can be obtained by ultrafiltration and ultracentrifugation in an equal quality for in vitro experiments. Especially for later therapeutic usage we recommend ultrafiltration due to a higher concentration without aggregation of extracellular vesicles in comparison to exosomes obtained by ultracentrifugation.
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Publication Date: 2019-01-28 PubMed ID: 30691449PubMed Central: PMC6348641DOI: 10.1186/s12917-019-1789-9Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This study looked at different techniques for isolating and identifying exosomes from equine adipose derived mesenchymal stem cells. The findings showed that exosomes can be acquired through ultracentrifugation, ultrafiltration, or a charge-based precipitation method. The derived particles were analysed with nanoparticle tracking, immunofluorescence staining and transmission electron microscopy, ultimately suggesting that ultrafiltration may be the most effective method for producing a high concentration of extracellular vesicles.

Explanation of the Research Article

  • The paper begins by acknowledging the role of mesenchymal stem cells in different therapeutic approaches, including treatments for osteoarthritis, tendon injuries, and bone defects. Researchers suggest that these stem cells’ curative effect might be due to a larger intercellular communication system, primarily facilitated by exosomes (small vesicles that transport proteins, lipids, and nucleic acids from one cell to another).

Experimental Methods and Results

  • Exploring the crucial but poorly understood role of exosomes required the researchers to first isolate and identify these vesicles from equine adipose derived mesenchymal stem cells. The study utilised three common techniques for this process: ultracentrifugation at 100,000g, ultrafiltration with 3 kDa exclusion membranes, and a charge-based precipitation method.
  • Their results showed that the mean sizes and amounts of exosomes varied with the technique used, varying from 116.2nm to 453.1nm in diameter and varying counts of particles per millilitre.

Identification of Exosomes

  • The researchers used immunofluorescence staining to examine the presence of tetraspanins CD9, CD63, and CD81 – recognised markers for exosomes – in the cellular product they isolated. They then utilised transmission electron microscopy and immunogold labelling with CD9 and CD90 to further investigate the morphology and marker presence of the exosomes.
  • The study also conducted a western blot analysis of CD9 and CD90 in the exosomes to ensure the specificity of the available antibodies against equine antigens.

Conclusions and Recommendations

  • The study concluded that exosomes derived from equine mesenchymal stem cells could be obtained through ultracentrifugation or ultrafiltration with equal quality for in vitro experiments. However, the authors recommend ultrafiltration as it provided a higher concentration of extracellular vesicles without aggregation – making it especially suitable for therapeutic applications.

Cite This Article

APA
Klymiuk MC, Balz N, Elashry MI, Heimann M, Wenisch S, Arnhold S. (2019). Exosomes isolation and identification from equine mesenchymal stem cells. BMC Vet Res, 15(1), 42. https://doi.org/10.1186/s12917-019-1789-9

Publication

BMC veterinary research
ISSN: 1746-6148
NlmUniqueID: 101249759
Country: England
Language: English
Volume: 15
Issue: 1
Pages: 42
PII: 42

Researcher Affiliations

Klymiuk, Michele Christian
  • Institute of Veterinary-Anatomy, -Histology and -Embryology, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, Frankfurter Strasse 98, Giessen, 35392, Germany. michele.klymiuk@vetmed.uni-giessen.de.
Balz, Natalie
  • Institute of Veterinary-Anatomy, -Histology and -Embryology, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, Frankfurter Strasse 98, Giessen, 35392, Germany.
Elashry, Mohamed I
  • Institute of Veterinary-Anatomy, -Histology and -Embryology, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, Frankfurter Strasse 98, Giessen, 35392, Germany.
  • Anatomy and Embryology Department, Faculty of Veterinary Medicine, University of Mansoura, Mansoura, 35516, Egypt.
Heimann, Manuela
  • Institute of Veterinary-Anatomy, -Histology and -Embryology, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, Frankfurter Strasse 98, Giessen, 35392, Germany.
Wenisch, Sabine
  • Clinic of Small Animals, c/o Institute of Veterinary-Anatomy, -Histology and -Embryology, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, Frankfurter Strasse 98, Giessen, 35392, Germany.
Arnhold, Stefan
  • Institute of Veterinary-Anatomy, -Histology and -Embryology, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, Frankfurter Strasse 98, Giessen, 35392, Germany.

MeSH Terms

  • Animals
  • Cytological Techniques / methods
  • Exosomes
  • Horses
  • Mesenchymal Stem Cells / metabolism
  • Ultrafiltration

Conflict of Interest Statement

AUTHORS’ INFORMATION: No additional information. ETHICS APPROVAL AND CONSENT TO PARTICIPATE: Samples were collected from slaughtered horses. Therefore, no ethical approval was necessary according to German laws to conduct this study. CONSENT FOR PUBLICATION: Not applicable. COMPETING INTERESTS: The authors declare that they have no competing interests. PUBLISHER’S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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