Adhesion Complex Formation in Cultivated Limbal Epithelium on Human Amniotic Membrane-In vitro Model

Abstract

Abstract: : Purpose: To investigate the adhesion complex formation in limbal epithelium after their cultivation on human amniotic membrane before transplantation of cultivated epithelium to limbal deficient model Methods: Limbal tissue from rabbit's cornea was cut into 2 mm²x 2mm², 100 um thickness and was treated with 1.2 U/ml of dispase for 30 minutes at 37¡É. Three pieces of explant were placed, epithelial side down, on amniotic membrane on a culture plate insert. Epithelial cells were cocultured with mitomycin C treated 3T3 fibroblasts. The culture medium used was Dulbecco's modified Eagle medium and Ham's F12 medium(1:1 mixture), and included fetal bovine serum(10%), insulin(5mg/ml), cholera toxin(0.1 nmol/l), epidermal growth factor(10ng/ml), and penicillin-streptomycin(50 IU/ml). Cultures were incubated at 37¡É in a carbon dioxide air incubator for up to 28 days, and the medium was changed every 2 days. Explants were left in the culture dish for the duration of the incubation. Air lifting was used after 2 weeks cultivation. Cultivated epithelium was fixed in 10% formaldehyde or in 1.0% glutaraldehyde at 2 weeks, 3 weeks and 4 weeks to observe the gross status of attachment and ultrastructures of hemidesmosome and anchoring fibril using light and transmission electron microscopy. Results: The cultivated corneal epithelium showed two to four layers of stratification and was well differentiated. Gross attachment of epithelium on amniotic membrane looked good under light microscopy. However, under electron microscopy, primitive pattern of hemidesmosome without any cytoplasmic intermediate filament was observed till 4 weeks. The density of hemidesmosome of cultivated rabbit epithelium was also lower than them of normal rabbit epithelium. Patches of anchoring fibril toward the stroma of amniotic membrane were found sporadically. Conclusions: The adhesion complex was incompletely formed in cultured limbal epithelium on amniotic membrane even after 4 weeks cultivation. This delayed formation of adhesion complex suggests cultivated epithelial cell may be susceptible to shed for some time when transplanted with amniotic membrane in vivo.

Keywords: cell adhesions/cell junctions • cornea: epithelium • microscopy: electron microscopy