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Takahiro Ogasawara, Masukazu INOIE, Miho FUJITA, Yuko SUWA, Yoko OUMI, Chikara SHINOHARA, Ken-ichiro HATA; Immunohistological Characterization Of Human-cultured-limbal Epithelium. Invest. Ophthalmol. Vis. Sci. 2011;52(14):297.
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© ARVO (1962-2015); The Authors (2016-present)
Evaluation of cytological characteristics of autologous human-cultured-limbal epithelium (HCLE) is necessary in order to demonstrate the its clinical efficacy. In this study, the cytological characteristics of HCLE were analyzed by transmission electron microscope (TEM) and immunostaining.
Human limbal epithelial cells isolated by trypsinization from human eye bank cornea were inoculated with X-irradiated 3T3-J2 feeder layer on culture flask. After cell cultivation at 37°C, 5% CO2, limbal epithelial cells, almost confluent, were subcultured on fibrin glue. The HCLE was produced with two passage cells culture for 6 days. The structure of them was analyzed by TEM. The formalin-fixed, paraffin embedded epithelia were produced and these cross-sections were used for hematoxylin-eosin staining and immunohistochemical analysis. The protein expressions such as Cytokeratin 3 (CK3) as a corneal epithelium marker, Cytokeratin 19 (CK19) levels as a conjunctival epithelium marker, MUC5AC as a goblet cells marker, p63 expression level as a stem cells marker, CD34 as a keratocytes marker, CD1a as a Langerhans cell marker, ZO-1, MUC1, MUC4 and MUC16 as a barrier function marker and Ki67 as a proliferating cell marker were analyzed.
The HCLE was multilayered to approximately 2- to 5-cell layers thick. Microvilli were observed on surface layer, and desmosome junction were observed between adjacent cells, but basement membrane were not. Ki67, CK3, CK19, p63, ZO-1, MUC1, MUC4 and MUC16 expressed on the HCLE were recognized, but CD1a, CD34 and MUC5AC were not.
The HCLE consists of multilayered epithelial cells without commingling of Langerhans cells and keratocytes, and has the characteristics of having proliferating cells and barrier functions including desmosome junction. These results show that the structure of the HCLE obtained with this culture technique resembles a normal corneal epithelial tissue. It is suggested that transplantation of this HCLE can be very useful for corneal epithelial defect. The basement membrane may be formed after interaction of corneal stroma with transplanted epithelium.
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