1Hernandez E., 1Meller D., 2Theiss C., 1Steuhl K.-P.

1Department of Ophthalmology, University of Essen, Essen, 2Department of Cytology, University of Bochum, Bochum

Objective: To determine if human limbal epithelial cells (HLEC) lack of gap junctions during ex vivo expansion on preserved human amniotic membrane (AM).
Methods: Primary human limbal (HLEC) and peripheral corneal (HPCEC) epithelial cells from limbal or peripheral corneal explants were cultured with SHEM on intact AM and compared to cultures on plastic. After 3 to 4 weeks, cell cultures were terminated. Development of gap junctions was analysed using mouse monoclonal antibodies to connexin 43 (Cx43) and evaluated by means of confocal laser microscopy. Corneal sections served as positive control. Functionality of gap junctions was evaluated by microinjection of Lucifer yellow into single cells using an pressure injection device under visual control with the aid of phase contrast optics.
Results: In vivo, expression of Cx43 was observed in corneal epithelial cells, whereas subpopulations of basal limbal cells were negative. In AM cultures, LE were noted to be almost negative for Cx43, but in PCE cultured on AM patchy-like expression of Cx43 was found. On plastic, LE showed an increased Cx43 expression compared to AM cultures. LE cultured on plastic exhibit a positive dye spread in 65% of injected cells whereas LE cultured on AM show a reduced dye spread index of 31%.
Conclusions: LE cultured on plastic are less undifferentiated and exhibit an increased formation of functional gap junctions whereas subpopulations of LE cultured on AM remain without gap junctions indicating that characteristics of limbal epithelial progenitor cells are preserved during ex vivo expansion on AM culture.

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