Culture morphology of the autologous cultivated corneal epithelium

• Published in: Klinika oczna Vol. 113; no. 7-9; p. 249
• Main Authors: Dobrowolski, Dariusz, Wylegała, Edward, Orzechowska-Wylegała, Bogusława, Wowra, Bogumił, Bielecka, Anna, Obuchowicz, Ewa, Małecki, Andrzej, Gabryel, Bozena
• Format: Journal Article
• Language: English
• Published: Poland 2011
• Subjects: Amnion - cytology; Cells, Cultured; Culture Media; Epithelial Cells - cytology; Epithelium, Corneal - cytology; Humans; Immunohistochemistry; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Tissue Culture Techniques - methods; Tissue Donors
• ISSN: 0023-2157

Ocular surgery based on cultivated corneal epithelium has become a very promising procedure eligible to restore the ocular surface. Analysis of morphologic features and the phenotype of cultivated epithelial cells determines their quality and eligibility of transplantation. Corneal epithelial cultures were carried out in 25 patients suffering from limbal deficiency after chemical or thermal burns. Fellow healthy eyes were the source of limbal epithelium for the culture. Limbal cells from a 2 mm2 biopsy were seeded on an amniotic membrane after enzymatic pretreatment. Cultures were carried in standard conditions in a supplemented DMEM HAM/F12 medium in the presence of 3T3 fibroblasts. Light microscopy was used to analyze the regularity of the cultivated epithelial layer, histologic examination was used to establish number of epithelial layers, and immunohistochemistry for epithelial and proliferation markers was applied to confirm cell origin and proliferative potential. Staining for cytokeratin 3, 12, 19, connexin 43, and protein p63 was performed. In 25 donors, 27 cultures of the epithelium were performed. In 2 cases, plates were contaminated. Both cultures were repeated. In 84% of the cultures, regular stratified growth of the epithelium with complete covering of amniotic membrane was observed. In 16% of cultures, growth was not regular, showing differences in the number of cell layers. Staining for cytokeratin 3/12 confirmed the corneal origin of cultivated epithelia. The number of epithelial layers ranged from 3 to 9; the average was 5.3 +/- 1.9 layers. Cultures of limbal epithelial cells are a valuable source of tissue for restoration of the corneal epithelium.