FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Home / Equine Research Bank / Vet Ophthalmol / Isolation and cultivation of equine corneal keratocytes, fib...
Veterinary ophthalmology2010; 13(1); 37-42; doi: 10.1111/j.1463-5224.2009.00755.x

Isolation and cultivation of equine corneal keratocytes, fibroblasts and myofibroblasts.

Abstract: To establish an in vitro model for the investigation of equine corneal wound healing. To accomplish this goal, a protocol to isolate and culture equine corneal keratocytes, fibroblasts and myofibroblasts was developed. ANIMAL MATERIAL: Equine corneal buttons were aseptically harvested from healthy research horses undergoing humane euthanasia for reasons unrelated to this study. Slit-lamp biomicroscopy was performed prior to euthanasia by a board-certified veterinary ophthalmologist to ensure that all samples were harvested from horses free of anterior segment disease. Methods: Equine corneal stroma was isolated using mechanical techniques and stromal sub-sections were then cultured. Customized media at different culture conditions was used to promote growth and differentiation of corneal stromal cells into keratocytes, fibroblasts and myofibroblasts. Results: Cell culture techniques were successfully used to establish a method for the isolation and culture of equine corneal keratocytes, fibroblasts and myofibroblasts. Immunohistochemical staining for alpha-smooth muscle and F-actin was used to definitively differentiate the three cell types. Conclusions: Equine corneal stromal keratocytes, fibroblasts and myofibroblasts can be predictably isolated and cultured in vitro using this protocol.
Get Full Text
Publication Date: 2010-02-13 PubMed ID: 20149174PubMed Central: PMC2930189DOI: 10.1111/j.1463-5224.2009.00755.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
  • Journal Article
  • Research Support
  • N.I.H.
  • Extramural
  • 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 establishes a method for isolating and cultivating three types of equine corneal cells – keratocytes, fibroblasts, and myofibroblasts – in a laboratory setting, which can be used to study wound healing in horse corneas.

Animal Material and Sample Collection

  • The team utilized corneal buttons, a type of tissue sample, from healthy research horses. These animals were being humanely euthanized for reasons unrelated to this study.
  • Before collecting samples, a slit-lamp biomicroscopy was conducted by a certified veterinary ophthalmologist to confirm that the horses were free from any anterior segment disease. This step ensured the reliability and accuracy of the test results.

Methods

  • The researchers began by mechanically isolating the equine corneal stroma, the dense, fibrous, connective tissue that forms the bulk of the cornea.
  • Sections of this stromal tissue were then cultured, or grown in a controlled environment in the lab.
  • Customized culture media under varied conditions was employed to encourage the growth and differentiation of the stromal cells into keratocytes, fibroblasts and myofibroblasts. These three types of cells play crucial roles in the wound-healing process.

Results

  • The custom-made cell culture techniques were effective in developing a consistent method to isolate and cultivate equine corneal keratocytes, fibroblasts, and myofibroblasts.
  • One way to confirm the successful differentiation of the cells into these three types was to apply immunohistochemical staining for alpha-smooth muscle actin and F-actin. These are proteins typically found in the respective cells and their presence serves as a marker for successful differentiation.

Conclusions

  • The research was successful in the predictable isolation and in vitro culture of equine corneal stromal keratocytes, fibroblasts, and myofibroblasts.
  • This accomplishment allows for further study in the field of corneal wound healing in horses. The ability to study these cells in a controlled laboratory setting can ultimately lead to advancements in treatment methods or the development of new therapeutic agents.

Cite This Article

APA
Buss DG, Giuliano EA, Sharma A, Mohan RR. (2010). Isolation and cultivation of equine corneal keratocytes, fibroblasts and myofibroblasts. Vet Ophthalmol, 13(1), 37-42. https://doi.org/10.1111/j.1463-5224.2009.00755.x

Publication

Veterinary ophthalmology
ISSN: 1463-5224
NlmUniqueID: 100887377
Country: England
Language: English
Volume: 13
Issue: 1
Pages: 37-42

Researcher Affiliations

Buss, Dylan G
  • College of Veterinary Medicine, University of Missouri, Columbia, MO, USA.
Giuliano, Elizabeth A
    Sharma, Ajay
      Mohan, Rajiv R

        MeSH Terms

        • Actins / metabolism
        • Animals
        • Cell Separation / veterinary
        • Cell Survival
        • Cells, Cultured
        • Cornea / cytology
        • Cornea / metabolism
        • Corneal Stroma / cytology
        • Corneal Stroma / metabolism
        • Fibroblasts / cytology
        • Horses / anatomy & histology
        • Polymerase Chain Reaction

        Grant Funding

        • I01 BX000357 / BLRD VA
        • R01 EY017294 / NEI NIH HHS
        • R01EY017294S1 / NEI NIH HHS
        • R01EY017294S2 / NEI NIH HHS

        References

        This article includes 27 references
        1. Barnett K. Color Atlas and Text of Equine Ophthalmology. Mosby-Wolfe; Baltimore: 1995.
        2. Gilger BC. Equine Ophthalmology. Elsevier Saunders; St. Louis: 2005.
        3. Gelatt KN. Veterinary Ophthalmology. 4. Blackwell Publishing; Ames: 2007.
        4. Jester JV, Barry-Lane PA, Cavanagh HD. Induction of alpha-smooth muscle actin expression and myofibroblast transformation in cultured corneal keratocytes. Cornea 1996;15:505–516.
          pubmed: 8862928
        5. Jester JV, Petroll WM, Cavanagh HD. Corneal stromal wound healing in refractive surgery: the role of myofibroblasts. Progress in Retinal and Eye Research 1999;18:311–356.
          pubmed: 10192516
        6. Masur SK, Dewal HS, Dinh TT. Myofibroblasts differentiate from fibroblasts when plated at low density. Proceedings of the National Academy of Sciences of the United States of America 1996;93:4219–4223.
          pmc: PMC39515pubmed: 8633044
        7. Wilson SE, Mohan RR, Ambrosio R Jr. The corneal wound healing response: cytokine-mediated interaction of the epithelium, stroma, and inflammatory cells. Progress in Retinal Eye Research 2001;20:625–637.
          pubmed: 11470453
        8. Netto MV, Mohan RR, Sinha S. Effect of prophylactic and therapeutic mitomycin C on corneal apoptosis, cellular proliferation, haze, and long-term keratocyte density in rabbits. Journal of Refractive Surgery 2006;22:562–574.
          pmc: PMC2756017pubmed: 16805119
        9. Leccisotti A. Mitomycin C in photorefractive keratectomy: effect on epithelialization and predictability. Cornea 2008;27:288–291.
          pubmed: 18362654
        10. Thornton I, Xu M, Krueger RR. Comparison of standard (0.02%) and low dose (0.002%) mitomycin C in the prevention of corneal haze following surface ablation for myopia. Journal of Refractive Surgery 2008;24:S68–S76.
          pubmed: 18269154
        11. Shalaby A, Kaye GB, Gimbel HV. Mitomycin C in photorefractive keratectomy. Journal of Refractive Surgery 2009;25:S93–S97.
          pubmed: 19248535
        12. Nassaralla BA, McLeod SD, Nassaralla JJ Jr. Prophylactic mitomycin C to inhibit corneal haze after photorefractive keratectomy for residual myopia following radial keratotomy. Journal of Refractive Surgery 2007;23:226–232.
          pubmed: 17385287
        13. Srinivasan S, Drake A, Herzig S. Photorefractive keratectomy with 0.02% mitomycin C for treatment of residual refractive errors after LASIK. Journal of Refractive Surgery 2008;24:S64–S67.
          pubmed: 18269153
        14. Sharma A, Mehan MM, Sinha S. Trichostatin A inhibits corneal haze in vitro and in vivo. Investigative Ophthalmology and Visual Science 2009;50:2695–2701.
          pmc: PMC3711114pubmed: 19168895
        15. Mohan RR, Stapleton WM, Sinha S. Decorin gene transfer into keratocytes inhibits alpha smooth muscle actin (SMA) expression and myofibroblast transformation. Investigative Ophthalmology & Visual Science 2006;47:E 1815.
        16. Barnett KC. Equine Ophthalmology: An Atlas and Text. 2. Saunders; Edinburgh; New York: 2004.
        17. Haber M, Cao Z, Panjwani N. Effects of growth factors (EGF, PDGF-BB and TGF-beta 1) on cultured equine epithelial cells and keratocytes: implications for wound healing. Veterinary Ophthalmology 2003;6:211–217.
          pubmed: 12950652
        18. Hong JW, Liu JJ, Lee JS. Proinflammatory chemokine induction in keratocytes and inflammatory cell infiltration into the cornea. Investigative Ophthalmology and Visual Science 2001;42:2795–2803.
          pubmed: 11687520
        19. Masur SK, Cheung JK, Antohi S. Identification of integrins in cultured corneal fibroblasts and in isolated keratocytes. Investigative Ophthalmology and Visual Science 1993;34:2690–2698.
          pubmed: 8344791
        20. Werner A, Braun M, Reichl S. Establishing and functional testing of a canine corneal construct. Veterinary Ophthalmology 2008;11:280–289.
          pubmed: 19046287
        21. Werner A, Braun M, Kietzmann M. Isolation and cultivation of canine corneal cells for in vitro studies on the anti-inflammatory effects of dexamethasone. Veterinary Ophthalmology 2008;11:67–74.
          pubmed: 18302570
        22. Toropainen E, Ranta VP, Talvitie A. Culture model of human corneal epithelium for prediction of ocular drug absorption. Investigative Ophthalmology and Visual Science 2001;42:2942–2948.
          pubmed: 11687540
        23. Tegtmeyer S, Reichl S, Muller-Goymann CC. Cultivation and characterization of a bovine in vitro model of the cornea. Pharmazie 2004;59:464–471.
          pubmed: 15248462
        24. Netto MV, Mohan RR, Ambrosio R Jr. Wound healing in the cornea: a review of refractive surgery complications and new prospects for therapy. Cornea 2005;24:509–522.
          pubmed: 15968154
        25. Netto MV, Mohan RR, Sinha S. Stromal haze, myofibroblasts, and surface irregularity after PRK. Experimental Eye Research 2006;82:788–797.
          pmc: PMC2693937pubmed: 16303127
        26. Mohan RR, Hutcheon AE, Choi R. Apoptosis, necrosis, proliferation, and myofibroblast generation in the stroma following LASIK and PRK. Experimental Eye Research 2003;76:71–87.
          pubmed: 12589777
        27. Jester JV, Moller-Pedersen T, Huang J. The cellular basis of corneal transparency: evidence for ‘corneal crystallins’. Journal of Cell Science 1999;112(Pt 5):613–622.
          pubmed: 9973596

        Citations

        This article has been cited 16 times.
        1. Sawicki S, Bugno-Poniewierska M, Żurowski J, Szmatoła T, Semik-Gurgul E, Bochenek M, Karnas E, Gurgul A. Comparative Transcriptome and MicroRNA Profiles of Equine Mesenchymal Stem Cells, Fibroblasts, and Their Extracellular Vesicles. Genes (Basel) 2025 Aug 5;16(8).
          doi: 10.3390/genes16080936pubmed: 40869984google scholar: lookup
        2. Surovtseva MA, Kim II, Bondarenko NA, Lykov AP, Krasner KY, Chepeleva EV, Bgatova NP, Trunov AN, Chernykh VV, Poveshchenko OV. Derivation of Human Corneal Keratocytes from ReLEx SMILE Lenticules for Cell Therapy and Tissue Engineering. Int J Mol Sci 2023 May 16;24(10).
          doi: 10.3390/ijms24108828pubmed: 37240176google scholar: lookup
        3. Nishtala K, Panigrahi T, Shetty R, Kumar D, Khamar P, Mohan RR, Deshpande V, Ghosh A. Quantitative Proteomics Reveals Molecular Network Driving Stromal Cell Differentiation: Implications for Corneal Wound Healing. Int J Mol Sci 2022 Feb 25;23(5).
          doi: 10.3390/ijms23052572pubmed: 35269714google scholar: lookup
        4. Surovtseva MA, Poveshchenko OV, Krasner KY, Kim II, Lykov AP, Bondarenko NA, Shul'mina LA, Trunov AN, Chernykh VV. Morphofunctional Properties of Corneal Stromal Cells. Bull Exp Biol Med 2021 Nov;172(1):96-99.
          doi: 10.1007/s10517-021-05339-5pubmed: 34791562google scholar: lookup
        5. Zhang Y, Liu Z, Wang K, Lu S, Fan S, Xu L, Cai B. Macrophage migration inhibitory factor regulates joint capsule fibrosis by promoting TGF-β1 production in fibroblasts. Int J Biol Sci 2021;17(7):1837-1850.
          doi: 10.7150/ijbs.57025pubmed: 33994866google scholar: lookup
        6. Shetty R, Kumar NR, Subramani M, Krishna L, Murugeswari P, Matalia H, Khamar P, Dadachanji ZV, Mohan RR, Ghosh A, Das D. Safety and efficacy of combination of suberoylamilide hydroxyamic acid and mitomycin C in reducing pro-fibrotic changes in human corneal epithelial cells. Sci Rep 2021 Feb 23;11(1):4392.
          doi: 10.1038/s41598-021-83881-ypubmed: 33623133google scholar: lookup
        7. Marlo TL, Giuliano EA, Tripathi R, Sharma A, Mohan RR. Altering equine corneal fibroblast differentiation through Smad gene transfer. Vet Ophthalmol 2018 Mar;21(2):132-139.
          doi: 10.1111/vop.12485pubmed: 28685927google scholar: lookup
        8. Sherman AB, Gilger BC, Berglund AK, Schnabel LV. Effect of bone marrow-derived mesenchymal stem cells and stem cell supernatant on equine corneal wound healing in vitro. Stem Cell Res Ther 2017 May 25;8(1):120.
          doi: 10.1186/s13287-017-0577-3pubmed: 28545510google scholar: lookup
        9. Gronkiewicz KM, Giuliano EA, Kuroki K, Bunyak F, Sharma A, Teixeira LB, Hamm CW, Mohan RR. Development of a novel in vivo corneal fibrosis model in the dog. Exp Eye Res 2016 Feb;143:75-88.
          doi: 10.1016/j.exer.2015.09.010pubmed: 26450656google scholar: lookup
        10. He R, Wang Z, Lu Y, Huang J, Ren J, Wang K. Chaperonin containing T-complex polypeptide subunit eta is a potential marker of joint contracture: an experimental study in the rat. Cell Stress Chaperones 2015 Nov;20(6):959-66.
          doi: 10.1007/s12192-015-0624-xpubmed: 26220476google scholar: lookup
        11. Fink MK, Giuliano EA, Tandon A, Mohan RR. Therapeutic potential of Pirfenidone for treating equine corneal scarring. Vet Ophthalmol 2015 May;18(3):242-50.
          doi: 10.1111/vop.12194pubmed: 25041235google scholar: lookup
        12. Das SK, Gupta I, Cho YK, Zhang X, Uehara H, Muddana SK, Bernhisel AA, Archer B, Ambati BK. Vimentin knockdown decreases corneal opacity. Invest Ophthalmol Vis Sci 2014 May 22;55(7):4030-40.
          doi: 10.1167/iovs.13-13494pubmed: 24854859google scholar: lookup
        13. Yang Y, Wang Z, Yang H, Wang L, Gillespie SR, Wolosin JM, Bernstein AM, Reinach PS. TRPV1 potentiates TGFβ-induction of corneal myofibroblast development through an oxidative stress-mediated p38-SMAD2 signaling loop. PLoS One 2013;8(10):e77300.
          doi: 10.1371/journal.pone.0077300pubmed: 24098582google scholar: lookup
        14. Fan TJ, Hu XZ, Zhao J, Niu Y, Zhao WZ, Yu MM, Ge Y. Establishment of an untransfected human corneal stromal cell line and its biocompatibility to acellular porcine corneal stroma. Int J Ophthalmol 2012;5(3):286-92.
          doi: 10.3980/j.issn.2222-3959.2012.03.07pubmed: 22773974google scholar: lookup
        15. Buss DG, Giuliano E, Sharma A, Mohan RR. Gene delivery in the equine cornea: a novel therapeutic strategy. Vet Ophthalmol 2010 Sep;13(5):301-6.
          doi: 10.1111/j.1463-5224.2010.00813.xpubmed: 20840107google scholar: lookup
        16. Buss DG, Sharma A, Giuliano EA, Mohan RR. Efficacy and safety of mitomycin C as an agent to treat corneal scarring in horses using an in vitro model. Vet Ophthalmol 2010 Jul;13(4):211-8.
          doi: 10.1111/j.1463-5224.2010.00782.xpubmed: 20618797google scholar: lookup
        Subscribe to our newsletter
        Join 99,344 horse owners like you who receive our email updates on horse health and nutrition.