98th Annual Meeting DOG 2000

V 616

Human amniotic membranes after different cryopreservation methods

F. Sistani1 , C. Erb1, D. Brockmann1, A. Nietgen1, A. Stan2, R. Winter1

Introduction: Human amniotic membranes are being used to reconstruct ocular surface. We examined the viability of amniotic epithelium after different cryopreservation methods. We introduce an enzymatic method to yield acellular amnotic membranes.

Material und Methods: Human amniotic membranes were incubated in MEM medium at – 80°C and –196°C with and without addition of Glycerol. After thawing the membranes the viability of the epithelium was assesed using trypan blue staining. For removal of the epithelium, the membranes were treated with Dispase II for 15 minutes at 37°C and were then washed in PBS solution.

Results: After cryopreservation of the membranes at – 80°C in pure MEM medium 75% of the epithelial cells were vital. After addition of glycerol to this group 65% of the cells survived cryopreservation. Cryopreserving the membranes in medium alone at –196°C led to a survival rate of 30%. After addition of glycerol, there were no viable cells detectable. Treating the membranes with Dispase II for 15 minutes at 37°C, epithelial cells could be completely removed

Conclusion: None of the above mentioned cryopreservation methods led to an acellular membrane. Supposing that the amniotic membranes promote reepithelialisation by just acting as a mechanical support for the ocular epithelium, it would be desirable to remove amniotic epithelium to decrease the possible inflammatry reactions to the foreign cells. The enzymatic digestion with Dispase II offers an easy way to achieve completely acellular amniotic membranes, without impairement of the elasticity of the membrane itself.

1Dept. of Ophthalmology and 2Dept. of Neuropathology, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany