Abstract
Purpose: :
To establish human conjunctival epithelial cell culture on amniotic membrane.
Methods: :
A conjunctival fragment of approximately 2x4mm was harvested from different living donors who underwent cataract or pterygium surgery. All donors signed a inform consent prior to the procedure. The conjunctival fragment was sent to the laboratory. Under sterile conditions, the tissue was divided into an anterior and a posterior portion. The anterior portion was divided into two fragments. One was cultivated on denuded human amniotic membrane, and the other was placed on a culture plate. The cultures were incubated with DMEM/HAM’S F12 medium at 37ºC and 5% CO2. The culture medium was changed 3 times a week for 3 weeks. After this period, the cultures were fixed for immunocytochemical analysis for epithelial cytokeratins (K3, K19, MUC5) and proliferation markers (Ki-67, p63). We also performed impression cytology, electron microscopy and confocal microscopy analysis.
Results: :
Conjunctival epithelial cells (n=4) expanded successfully either on culture plate or amniotic membrane. Impression cytology demonstrated the presence of compact conjunctival epithelium and goblet cells. Immunocytochemical analysis showed positivity for K3, K19, MUC5 and 20 to 30 % positivity for Ki-67 and p63.
Conclusions: :
Our results demonstrated that it is possible to cultivate human conjunctival epithelial and goblet cells ex vivo on human amniotic membrane. This method may represent an important step to be used in the treatment of many ocular surface diseases.
Keywords: conjunctiva • cell survival • microscopy: light/fluorescence/immunohistochemistry