Tissue Engineering To Cultivate Human Limbal Epithelial Cells

Objective To cultivate human limbal epithelial cells in vitro and investigate the growth of cultured cells. Limbal epithelial cells were cultured on denuded human amniotic membrane and biological amniotic membrane as substrates in vitro for 28d. To investigate the growth, the stratification and the adhesion complex formation of cultured cells on the substrates. To evaluate biological amniotic membrane is a potentially suitable substrate to culture limbal epithelial cells.Methods Limbal epithelial cells were cultured with cell-suspension technique and explant technique. Inverted microscope was performed to observe the growth of the cultured cells. Immunohistochemistry for K3 and p63 were used to identify the limbal stem cells which had proliferating potential. Denuded amniotic membrane and biological amniotic membrane were used as substrates to cultivate limbal epithelial cells with both cell-suspension and explant culture methods. Hematoxylin staining, HE staining and transmission electromicroscope were performed to observe the cultured cells.Results Both cell-suspension and explant culture methods produced healthy epithelial cell layers. The explant culture had higher achievement ratio than cell-suspension culture. On 14d a few cells stained negatively to K3 antibody and positively to p63 antibody. Limbal epithelial cells cultivated on both denuded amniotic and biological amniotic formed nicely three to four cell thick stratified layers. The typical hemidesmosomal junctions were found between biological amniotic membrane and the neighboring basal cells on 28d.Conclusions Both cell-suspension and explant culture methods produced healthy epithelial cell layers which contained limbal stem cells. The explant culture had higher achievement ratio than cell-suspension culture. Biological amniotic membrane is a suitable substrate to culture limbal epithelial cells.