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Published on: July 16, 2013
Comparison of techniques for corneal epithelium cell culture for the collection of conditioned medium
Magda Massae Hata Viveiros1, Luís Henrique Zucoloto2, Álvio Issao Shiguematsu1
1Department of Ophthalmology, Faculdade de Medicina de Botucatu, Universidade Estadual Paulista "Júlio de Mesquita Filho," Botucatu, SP, Brazil.
Purposes:
To determine the best protocol in obtaining the higher yield of conditioned culture medium to be used for the bone marrow mesenchymal stem cell differentiation into corneal epithelial cells, five techniques for the primary culture of human corneal epithelial cells were evaluated.
Methods:
The studied culture techniques of corneal epithelial cells were: explants in culture flasks with and without hydrophilic surface treatment, on amniotic membrane, with enzymatic digestion, and by corneal scraping. The conditioned culture medium collected from these cultures was used to differentiate human bone marrow mesenchymal stem cells into corneal epithelial cells, which were characterized using flow cytometry with pan-cytokeratin and the corneal-specific markers, cytokeratin 3 and cytokeratin 12.
Results:
The culture technique using flasks with hydrophilic surface treatment resulted in the highest yield of conditioned culture medium. Flasks without surface treatment resulted to a very low success rate. Enzymatic digestion and corneal scraping showed contamination with corneal fibroblasts. The culture on amniotic membranes only allowed the collection of culture medium during the 1st cell confluence. The effectiveness of cell differentiation was confirmed by cytometry analysis using the collected conditioned culture medium, as demonstrated by the expressions of cytokeratin 3 (95.3%), cytokeratin 12 (93.4%), and pan-cytokeratin (95.3%).
Conclusion:
The culture of corneal epithelial cell explants in flasks with hydrophilic surface treatment is the best technique for collecting a higher yield of conditioned culture medium to be used to differentiate mesenchymal stem cells.
