Effect of limbal explant orientation on the histology, phenotype, ultrastructure and barrier function of cultured limbal epithelial cells.
OBJECTIVE
To compare the histology, phenotype, ultrastructure and barrier function of cultured limbal epithelial cells using two explant culture protocols.


METHODS
Epithelial cells were cultured for 16 days from limbal explants, positioned with either the stromal side (stromal group) or the epithelial side (epithelial group) on intact amniotic membranes. The cultured epithelium (n = 56) was examined using light microscopy, immunohistochemistry for K3, Cx43, ABCG2 and p63 expression, Western blot analysis of DeltaNp63alpha, transmission electron microscopy, a horseradish peroxidase (HRP) permeability assay and scanning electron microscopy.


RESULTS
The epithelial group demonstrated a significantly higher expression of p63-positive cells (85.7 +/- 4.2%) than the stromal group (75.3 +/- 8.9%), and Western blots showed a stronger band of DeltaNp63alpha. K3 and ABCG2 were not detected in either group, whereas Cx43 displayed moderate immunostaining in the suprabasal layer. The number of cell layers, the desmosome number and the undulation length in the epithelial group were not significantly different from those in the stromal group. In both groups, HRP accumulated on the apical surface of the superficial cells, and scanning electron microscopy demonstrated tightly apposed superficial cells.


CONCLUSIONS
Our findings indicate that limbal explants positioned epithelial side down may give rise to cultured epithelia with higher expression of p63 and DeltaNp63alpha.
Raeder
Sten
S
Centre for Eye Research, Department of Ophthalmology, Ullevål University Hospital, University of Oslo, Oslo, Norway. sten.rader@medisin.uio.no
Utheim
Tor Paaske
TP
Utheim
Oygunn Aass
OA
Cai
Yiqing
Y
Roald
Borghild
B
Lyberg
Torstein
T
Kjeldsen-Kragh
Jens
J
Ramstad
Hanne
H
Messelt
Edvard
E
Nicolaissen
Bjørn
B
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000328:Adult; D000650:Amnion; D001692:Biological Transport / Q000502:physiology; D015153:Blotting, Western; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods; D018031:Connexin 43 / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology* / Q000502:physiology* / Q000648:ultrastructure; D006801:Humans; D007124:Immunoenzyme Techniques; D053558:Keratin-3 / Q000378:metabolism; D016850:Limbus Corneae / Q000166:cytology*; D008565:Membrane Proteins / Q000378:metabolism; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000378:metabolism; D010641:Phenotype; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins / Q000378:metabolism
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; C081876:CKAP4 protein, human; D018031:Connexin 43; D004268:DNA-Binding Proteins; C479360:KRT3 protein, human; D053558:Keratin-3; D008565:Membrane Proteins; D009363:Neoplasm Proteins; C115051:TP63 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins