V481

Transplantation of preserved anmiotic membrane (AMT) is a new method for ocular surface reconstrution. Maintainance of the biological integrity of the membrane is a prerequisite for stable reepithelialisation after surgery. As a first step to examine the biological mechanisms underlying reepithelialization of the membrane, we describe the surface of the membrane priot to and after kryoconservation.

Methods: Amniotic membrane was peeled off the placenta and fixated on special polystyrol carriers. Membranes were fixated in increasing concentrations of glycerol (0-50% in DMEM) and preserved at -80ºC. After thawing membranes were embedded for frozen and paraffin sections. Vital staining of the epithelial cells was performed on native membranes and after kryoconservation using alcian red and trypan blue. Vital staining was also performed with trypsinized cells. The proliferative capacity of amniotic membrane epithelial cells was investigated in cell culture (DMEM+ 10% FBS).

Results: Native amniotic membrane is covered by by a single layer of epithelial cells. Within the first hours after cesarean section cells in situ and after trypsination were vital. In addition they were able to proliferate in cell culture. After freezing in up to 50% glycerol cells remained in situ but were not vital. Upon culture we could not detect cell proliferation but rather a progressive reduction of the number of cells on the surface of the membrane.

Discussion: Interestingly amniotic membrane epithelial cells persist even after kryopreservation but these cells are not viable. The presence of these cells following AMT offers a possible mechanism whereby boung proteins such as cytokines, adhesion molecules or proteinase inhibitors facilitate migration and adhesion of corneal epithelial cells as well as proliferation of stromal keratocytes.

Department of Ophthalmology University of Heidelberg, INF 400, D-69120 Heidelberg