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Home / Equine Research Bank / Equine Vet J / Isolation of equine bone marrow-derived mesenchymal stem cel...
Equine veterinary journal2010; 42(6); 519-527; doi: 10.1111/j.2042-3306.2010.00098.x

Isolation of equine bone marrow-derived mesenchymal stem cells: a comparison between three protocols.

Abstract: There is a need to assess and standardise equine bone marrow (BM) mesenchymal stem cell (MSC) isolation protocols in order to permit valid comparisons between therapeutic trials at different sites. Objective: To compare 3 protocols of equine BM MSC isolation: adherence to a plastic culture dish (Classic) and 2 gradient density separation protocols (Percoll and Ficoll). Methods: BM aspirates were harvested from the sternum of 6 mares and MSCs isolated by all 3 protocols. The cell viability after isolation, MSC yield, number of MSCs attained after 14 days of culture and the functional characteristics (self-renewal (CFU) and multilineage differentiation capacity) were determined for all 3 protocols. Results: The mean +/- s.d. MSC yield from the Percoll protocol was significantly higher (6.8 +/- 3.8%) than the Classic protocol (1.3 +/- 0.7%). The numbers of MSCs recovered after 14 days culture per 10 ml BM sample were 24.0 +/- 12.1, 14.6 +/- 9.5 and 4.1 +/- 2.5 x 10(6) for the Percoll, Ficoll and Classic protocols, respectively, significantly higher for the Percoll compared with the Classic protocol. Importantly, no significant difference in cell viability or in osteogenic or chondrogenic differentiation was identified between the protocols. At Passage 0, cells retrieved with the Ficoll protocol had lower self-renewal capacity when compared with the Classic protocol but there was no significant difference between protocols at Passage 1. There were no significant differences between the 3 protocols for the global frequencies of CFUs at Passage 0 or 1. Conclusions: These data suggest that the Percoll gradient density separation protocol was the best in terms of MSC yield and self-renewal potential of the MSCs retrieved and that MSCs retrieved with the Ficoll protocol had the lowest self-renewal but only at passage 0. Then, the 3 protocols were equivalent. However, the Percoll protocol should be considered for equine MSC isolation to minimise culture time.
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Publication Date: 2010-08-19 PubMed ID: 20716192DOI: 10.1111/j.2042-3306.2010.00098.xGoogle Scholar: Lookup
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  • Comparative Study
  • Journal Article
  • Research Support
  • Non-U.S. Gov't

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 research compares three methods of isolating equine bone marrow-derived mesenchymal stem cells. It found that a method known as the Percoll gradient density separation protocol produced the highest yield and the most self-renewing cells, compared to the Classic and Ficoll protocols.

Objective of the Research

  • The study was intended to assess and standardize isolation protocols of eqine bone marrow (BM) mesenchymal stem cells (MSCs). This is important to allow meaningful comparisons between therapeutic trials conducted in different places.

Methodology

  • Three isolation protocols were compared – the Classic method, which relies on the stem cells adhering to the plastic surface of a culture dish, and two types of gradient density separation protocols, Percoll and Ficoll.
  • Bone marrow was drawn from six horses and the MSCs were then isolated using each of the three protocols.
  • Various factors were then considered to determine the effectiveness of each method. These included the viability of the cells immediately after isolation, the yield of MSCs, the number of MSCs produced after two weeks of culture, and their functional characteristics. The last of these focused on their capacity for self-renewal and ability to differentiate into various cell types.

Results and Conclusion

  • The Percoll protocol yielded significantly more mesenchymal stem cells (averaging 6.8%) than the Classic protocol (averaging 1.3%).
  • After 14 days, significantly more MSCs were harvested using the Percoll protocol compared to the Classic and Ficoll methods.
  • There was no notable difference in cell viability or the ability to differentiate into bone or cartilage cells among the cells isolated by the three protocols.
  • Cells obtained through the Ficoll protocol showed lower self-renewal capacity at Passage 0 (immediately after isolation) in comparison with those isolated using the Classic protocol. However, by Passage 1 (after one round of cell growth and reproduction), there was no significant difference among the three protocols.
  • The researchers concluded that the Percoll gradient density separation protocol was the most effective for isolating equine mesenchymal stem cells in terms of yield and self-renewing abilities. They also noted that the Percoll protocol could minimize the time required for MSC culture. Therefore, this method should be the preferential choice for future isolation of equine MSC.

Cite This Article

APA
Bourzac C, Smith LC, Vincent P, Beauchamp G, Lavoie JP, Laverty S. (2010). Isolation of equine bone marrow-derived mesenchymal stem cells: a comparison between three protocols. Equine Vet J, 42(6), 519-527. https://doi.org/10.1111/j.2042-3306.2010.00098.x

Publication

Equine veterinary journal
ISSN: 0425-1644
NlmUniqueID: 0173320
Country: United States
Language: English
Volume: 42
Issue: 6
Pages: 519-527

Researcher Affiliations

Bourzac, C
  • Département de Sciences Cliniques, Faculté de Médecine Vétérinaire, Université de Montréal, Québec, Canada.
Smith, L C
    Vincent, P
      Beauchamp, G
        Lavoie, J-P
          Laverty, S

            MeSH Terms

            • Adipocytes / cytology
            • Adipocytes / physiology
            • Animals
            • Bone Marrow Cells / cytology
            • Bone Marrow Cells / physiology
            • Cartilage / cytology
            • Cell Culture Techniques / methods
            • Cell Culture Techniques / veterinary
            • Cell Differentiation
            • Colony-Forming Units Assay
            • Horses
            • Mesenchymal Stem Cells / cytology
            • Mesenchymal Stem Cells / physiology

            Grant Funding

            • Canadian Institutes of Health Research

            Citations

            This article has been cited 26 times.
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