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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.
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Publication Date: 2010-02-13 PubMed ID: 20149174PubMed Central: PMC2930189DOI: 10.1111/j.1463-5224.2009.00755.xGoogle Scholar: Lookup
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  • 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

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        Citations

        This article has been cited 16 times.
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