Cryopreserved human amniotic membrane for ocular surface reconstruction

Background: Amniotic membrane transplantation is used for the reconstructio n of the ocular surface in the context of, for example, corneal ulcers or c onjunctival scarring. The mechanisms by which preserved amniotic membrane g rafts promote reepithelialization are unknown. As a first step the viabilit y and proliferative capacity of amnion cells following cryopreservation of membranes in glycerol is investigated. Methods: Fresh and cryopreserved (in 50% glycerol) amniotic membranes were investigated histologically and by v ital stains. Following enzymatic digestion, amniotic cells were stained for viability and cultured in DMEM+10% FBS. In addition, explant cultures were established from fresh and cryopreserved membranes. Results: Histologicacl examination showed no significant morphological alteration following cryop reservation. While fresh membranes contained predominantly vital cells, no such cells were detected following cryopreservation. Also, cells removed en zymatically from cryopreserved membranes were not viable and did not grow i n culture. While both epithelial and fibroblastic cells grew from fresh mem branes, no growth was seen from cryopreserved membranes, Conclusion: The re sults suggest that the technique for preservation which is most widely used for ophthalmological amniotic membrane transplantation significantly impai rs viability and proliferative capacity. This supports the clinical finding that neither immunological reactions nor signs of ingrowth of amniotic cel ls are observed in patients. Furthermore amniotic membrane grafts seem to f unction primarily as matrix and not by virtue of transplanted functional ce lls.