FREE SHIPPING over $40
OVER 10000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
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.
Get Full Text
Publication Date: 2010-08-19 PubMed ID: 20716192DOI: 10.1111/j.2042-3306.2010.00098.xGoogle Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • 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.
            1. Mayet A, Zablotski Y, Roth SP, Brehm W, Troillet A. Systematic review and meta-analysis of positive long-term effects after intra-articular administration of orthobiologic therapeutics in horses with naturally occurring osteoarthritis. Front Vet Sci 2023;10:1125695.
              doi: 10.3389/fvets.2023.1125695pubmed: 36908512google scholar: lookup
            2. Jammes M, Contentin R, Cassé F, Galéra P. Equine osteoarthritis: Strategies to enhance mesenchymal stromal cell-based acellular therapies. Front Vet Sci 2023;10:1115774.
              doi: 10.3389/fvets.2023.1115774pubmed: 36846261google scholar: lookup
            3. Yan X, Yan F, Mohammed HAG, Liu O. Maxillofacial-Derived Mesenchymal Stem Cells: Characteristics and Progress in Tissue Regeneration. Stem Cells Int 2021;2021:5516521.
              doi: 10.1155/2021/5516521pubmed: 34426741google scholar: lookup
            4. Chen YR, Yan X, Yuan FZ, Ye J, Xu BB, Zhou ZX, Mao ZM, Guan J, Song YF, Sun ZW, Wang XJ, Chen ZY, Wang DY, Fan BS, Yang M, Song ST, Jiang D, Yu JK. The Use of Peripheral Blood-Derived Stem Cells for Cartilage Repair and Regeneration In Vivo: A Review. Front Pharmacol 2020;11:404.
              doi: 10.3389/fphar.2020.00404pubmed: 32308625google scholar: lookup
            5. Tomlinson JE, Jager M, Struzyna A, Laverack M, Fortier LA, Dubovi E, Foil LD, Burbelo PD, Divers TJ, Van de Walle GR. Tropism, pathology, and transmission of equine parvovirus-hepatitis. Emerg Microbes Infect 2020;9(1):651-663.
              doi: 10.1080/22221751.2020.1741326pubmed: 32192415google scholar: lookup
            6. Al Naem M, Bourebaba L, Kucharczyk K, Röcken M, Marycz K. Therapeutic mesenchymal stromal stem cells: Isolation, characterization and role in equine regenerative medicine and metabolic disorders. Stem Cell Rev Rep 2020 Apr;16(2):301-322.
              doi: 10.1007/s12015-019-09932-0pubmed: 31797146google scholar: lookup
            7. Yang Q, Pinto VMR, Duan W, Paxton EE, Dessauer JH, Ryan W, Lopez MJ. In vitro Characteristics of Heterogeneous Equine Hoof Progenitor Cell Isolates. Front Bioeng Biotechnol 2019;7:155.
              doi: 10.3389/fbioe.2019.00155pubmed: 31355191google scholar: lookup
            8. Zahedi M, Parham A, Dehghani H, Kazemi Mehrjerdi H. Equine bone marrow-derived mesenchymal stem cells: optimization of cell density in primary culture. Stem Cell Investig 2018;5:31.
              doi: 10.21037/sci.2018.09.01pubmed: 30498742google scholar: lookup
            9. Rink BE, Beyer T, French HM, Watson E, Aurich C, Donadeu FX. The Fate of Autologous Endometrial Mesenchymal Stromal Cells After Application in the Healthy Equine Uterus. Stem Cells Dev 2018 Aug 1;27(15):1046-1052.
              doi: 10.1089/scd.2018.0056pubmed: 29790424google scholar: lookup
            10. Tsuru M, Ono A, Umeyama H, Takeuchi M, Nagata K. Ubiquitin-dependent proteolysis of CXCL7 leads to posterior longitudinal ligament ossification. PLoS One 2018;13(5):e0196204.
              doi: 10.1371/journal.pone.0196204pubmed: 29782494google scholar: lookup
            11. Schröck C, Eydt C, Geburek F, Kaiser L, Päbst F, Burk J, Pfarrer C, Staszyk C. Bone marrow-derived multipotent mesenchymal stromal cells from horses after euthanasia. Vet Med Sci 2017 Nov;3(4):239-251.
              doi: 10.1002/vms3.74pubmed: 29152317google scholar: lookup
            12. Eydt C, Geburek F, Schröck C, Hambruch N, Rohn K, Pfarrer C, Staszyk C. Sternal bone marrow derived equine multipotent mesenchymal stromal cells (MSCs): investigations considering the sampling site and the use of different culture media. Vet Med Sci 2016 Aug;2(3):200-210.
              doi: 10.1002/vms3.36pubmed: 29067195google scholar: lookup
            13. Rink BE, Amilon KR, Esteves CL, French HM, Watson E, Aurich C, Donadeu FX. Isolation and characterization of equine endometrial mesenchymal stromal cells. Stem Cell Res Ther 2017 Jul 12;8(1):166.
              doi: 10.1186/s13287-017-0616-0pubmed: 28701175google scholar: lookup
            14. Zahedi M, Parham A, Dehghani H, Mehrjerdi HK. Stemness Signature of Equine Marrow-derived Mesenchymal Stem Cells. Int J Stem Cells 2017 May 30;10(1):93-102.
              doi: 10.15283/ijsc16036pubmed: 28222255google scholar: lookup
            15. Williams LB, Co C, Koenig JB, Tse C, Lindsay E, Koch TG. Response to Intravenous Allogeneic Equine Cord Blood-Derived Mesenchymal Stromal Cells Administered from Chilled or Frozen State in Serum and Protein-Free Media. Front Vet Sci 2016;3:56.
              doi: 10.3389/fvets.2016.00056pubmed: 27500136google scholar: lookup
            16. Hopper N, Henson F, Brooks R, Ali E, Rushton N, Wardale J. Peripheral blood derived mononuclear cells enhance osteoarthritic human chondrocyte migration. Arthritis Res Ther 2015 Aug 7;17(1):199.
              doi: 10.1186/s13075-015-0709-zpubmed: 26249339google scholar: lookup
            17. Lin C, Shen M, Chen W, Li X, Luo D, Cai J, Yang Y. Isolation and purification of rabbit mesenchymal stem cells using an optimized protocol. In Vitro Cell Dev Biol Anim 2015 Nov;51(10):1102-8.
              doi: 10.1007/s11626-015-9933-8pubmed: 26202303google scholar: lookup
            18. Beane OS, Fonseca VC, Cooper LL, Koren G, Darling EM. Impact of aging on the regenerative properties of bone marrow-, muscle-, and adipose-derived mesenchymal stem/stromal cells. PLoS One 2014;9(12):e115963.
              doi: 10.1371/journal.pone.0115963pubmed: 25541697google scholar: lookup
            19. Gittel C, Brehm W, Burk J, Juelke H, Staszyk C, Ribitsch I. Isolation of equine multipotent mesenchymal stromal cells by enzymatic tissue digestion or explant technique: comparison of cellular properties. BMC Vet Res 2013 Oct 29;9:221.
              doi: 10.1186/1746-6148-9-221pubmed: 24168625google scholar: lookup
            20. Wee AS, Lim CK, Merican AM, Ahmad TS, Kamarul T. Total cell pooling in vitro: an effective isolation method for bone marrow-derived multipotent stromal cells. In Vitro Cell Dev Biol Anim 2013 Jun;49(6):424-32.
              doi: 10.1007/s11626-013-9626-0pubmed: 23708918google scholar: lookup
            21. McD￮ LA. Comparison of isolation and expansion techniques for equine osteogenic progenitor cells from periosteal tissue. Can J Vet Res 2012 Apr;76(2):91-8.
              pubmed: 23024451
            22. Delling U, Lindner K, Ribitsch I, Jülke H, Brehm W. Comparison of bone marrow aspiration at the sternum and the tuber coxae in middle-aged horses. Can J Vet Res 2012 Jan;76(1):52-6.
              pubmed: 22754095
            23. Xu Z, Yan L, Ge Y, Zhang Q, Yang N, Zhang M, Zhao Y, Sun P, Gao J, Tao Z, Yang Z. Effect of the calcium sensing receptor on rat bone marrow-derived mesenchymal stem cell proliferation through the ERK1/2 pathway. Mol Biol Rep 2012 Jul;39(7):7271-9.
              doi: 10.1007/s11033-012-1557-4pubmed: 22314915google scholar: lookup
            24. Mensing N, Gasse H, Hambruch N, Haeger JD, Pfarrer C, Staszyk C. Isolation and characterization of multipotent mesenchymal stromal cells from the gingiva and the periodontal ligament of the horse. BMC Vet Res 2011 Aug 2;7:42.
              doi: 10.1186/1746-6148-7-42pubmed: 21810270google scholar: lookup
            25. Khatibzadeh SM, Dahlgren LA, Caswell CC, Ducker WA, Werre SR, Bogers SH. Equine bone marrow-derived mesenchymal stromal cells reduce established S. aureus and E. coli biofilm matrix in vitro. PLoS One 2024;19(10):e0312917.
              doi: 10.1371/journal.pone.0312917pubmed: 39480794google scholar: lookup
            26. Yang GD, Ma DS, Ma CY, Bai Y. Research Progress on Cardiac Tissue Construction of Mesenchymal Stem Cells for Myocardial Infarction. Curr Stem Cell Res Ther 2024;19(7):942-958.
              doi: 10.2174/1574888X18666230823091017pubmed: 37612870google scholar: lookup
            Subscribe to our newsletter
            Join 99,727 horse owners like you who receive our email updates on horse health and nutrition.