V480

Transplantation of corneal epithelium e.g. in the context of limbal stem cell deficiency has until now been precluded by the lack of a suitable substratum. Amniotic membrane has been shown to be overgrown by epithelium when grafted onto corneas with epithelial defects. We therefore investigated the use of amniotic membrane for corneal epithelial transplantation.

Method: Amniotic membrane was prepared following cesarian deliveries and fixed onto specially cut polystyrol carriers prior to being stored at -80ºC in 50% DMEM/gycerol. Corneal epithelial cells from a cell line as well as from donor eyes (isolated by trephanation) were used. These cells were grown in coculture with a feeder layer of 3T3 cells in DMEM + 10% FBS. Epithelial growth was observed with phase contrast microscope on the surface of amniotic membranes at the air liquid interphase.

Results: We observed good attachment of epithelial cells to amniotic membranes. Histologically these cells grew in confluent multilayers with and without 3T3 cells. This was in contrast to culture on plastic where corneal epithelial cells can only form a single layer even in the presence of 3T3 cells.

Discussion: Preserved amniotic membrane can be used as a matrix to culture human corneal epithelium in vitro. In combination with a feeder layer which can secrete antiapoptotic factors epithelial cells from a patient can be expanded in culture. Thereafter these cells can be grafted onto the eye. Besides possible clinical application such a model can serve for investigation of the cell biology of the corneal epithelium.

Augenklinik der Universität Heidelberg, INF 400, D-69120 Heidelberg