title: Effect of limbal explant orientation on the histology, phenotype, ultrastructure and barrier function of cultured limbal epithelial cells.
abstract: 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.
authors.affiliation: Centre for Eye Research, Department of Ophthalmology, Ullevål University Hospital, University of Oslo, Oslo, Norway. sten.rader@medisin.uio.no
authors.forename: Sten
authors.initials: S
authors.lastname: Raeder
authors.forename: Tor Paaske
authors.initials: TP
authors.lastname: Utheim
authors.forename: Oygunn Aass
authors.initials: OA
authors.lastname: Utheim
authors.forename: Yiqing
authors.initials: Y
authors.lastname: Cai
authors.forename: Borghild
authors.initials: B
authors.lastname: Roald
authors.forename: Torstein
authors.initials: T
authors.lastname: Lyberg
authors.forename: Jens
authors.initials: J
authors.lastname: Kjeldsen-Kragh
authors.forename: Hanne
authors.initials: H
authors.lastname: Ramstad
authors.forename: Edvard
authors.initials: E
authors.lastname: Messelt
authors.forename: Bjørn
authors.initials: B
authors.lastname: Nicolaissen
mesh_terms: 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
chemical_list: 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