Corneal epithelial cultures generated from organ-cultured limbal tissue: factors influencing epithelial cell growth.
OBJECTIVE
To explore the in vitro proliferative potential of human limbal epithelial cells after 31 degrees C organ-culture storage and to investigate putative factors influencing it.


METHODS
185 cultures of limbal explants were carried-out either from full-thickness explants (n = 102) or from enzymatically dissociated cells (n = 83) seeded on a feeder layer of human keratocytes. Epithelial outgrowth was assessed by phase contrast microscopy using a computerized image analysis software. Cell phenotype was evaluated by transmission electron microscopy and immunocytology. Univariate and multivariate analysis were performed to determine factors influencing epithelial growth in culture.


RESULTS
An epithelial outgrowth of 100 square mm or more was observed in 52% of cultures, (average growth area: 440 +/- 256 mm at three weeks). Corneal epithelial phenotype was confirmed by transmission electron microscopy, and cytokeratin pattern. Cytokeratine 19, deltaNp63, nestin and vimentin positive staining revealed undifferentiated epithelial cells in both explant and cell suspension cultures at three weeks. Short death to cornea retrieval time (p < 0.03) and female donors (p < 0.01) were associated with higher cell growth. Enzymatic treatment of explants by trypsin, but not dispase, decreased cell proliferation at two (p < 0.03) and three weeks (p < 0.04). Donor age, duration of corneal storage, and source of the explant did not influence the cell growth.


CONCLUSIONS
Organ-culture conditions can preserve limbal cell mitotic potential if limbal tissue is excised early after circulatory arrest. Human keratocytes can be used as a feeder layer allowing epithelial cells to maintain poorly differentiated phenotype in culture. Further investigations are needed to explain the influence of the donor sex on epithelial cell growth in culture.
Zito-Abbad
Eleonora
E
Cornea Bank of EFS-Ile-de-France and Department of Ophthalmology, Centre Hospitalier National d'Ophtalmologie des XV-XX, Paris VI University, 28 rue de Charenton, 75012 Paris, France. eleonora.zito@noos.fr
Borderie
Vincent M
VM
Baudrimont
Marie
M
Bourcier
Tristan
T
Laroche
Laurent
L
Chapel
Catherine
C
Uzel
Jean-Louis
JL
D000368:Aged; D000369:Aged, 80 and over; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D004268:DNA-Binding Proteins / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology* / Q000502:physiology; D019573:Epithelium, Corneal / Q000166:cytology*; D005260:Female; D006801:Humans; D007381:Intermediate Filament Proteins / Q000378:metabolism; D007633:Keratins / Q000378:metabolism; D016850:Limbus Corneae / Q000166:cytology*; D008297:Male; D008858:Microscopy, Phase-Contrast; D008875:Middle Aged; D009419:Nerve Tissue Proteins / Q000378:metabolism; D064231:Nestin; D009924:Organ Culture Techniques; D010641:Phenotype; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism
D004268:DNA-Binding Proteins; D007381:Intermediate Filament Proteins; C577484:NES protein, human; D009419:Nerve Tissue Proteins; D064231:Nestin; C115051:TP63 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D014746:Vimentin; D007633:Keratins