title: Human epithelial cell cultures from superficial limbal explants.
abstract: OBJECTIVE
  To study the kinetics of growth and the phenotype of cells cultured from human limbal explants in a cholera toxin-free medium with no feeder cell layer.
  
  
  METHODS
  Human organ-cultured corneas were used to prepare limbal explants (full-thickness and superficial limbal explants) and corneal stromal explants. Cell growth kinetics and phenotypes were assessed by cultivating explants in cholera toxin-free Green medium. Epithelial and progenitor cell markers were assessed by immunocytochemistry, flow cytometry, and Reverse Transcription and Polymerase Chain Reaction (RT-PCR).
  
  
  RESULTS
  The successful epithelial cell growth rates from full thickness limbal explant and superficial limbal explant tissues were 41 and 86%, respectively (p=0.0001). The mean cell area and the percentage of small cells in superficial and full-thickness explant cultures were, respectively, 317 µm(2) and 429 µm(2), and 8.9% and 1.7% (p<0.001). The percentage of positive cells in superficial and full-thickness limbal explant cultures as assessed by immunocytochemistry were the following: broad spectrum cytokeratins (cytokeratins 4, 5, 6, 8, 10, 13, and 18 [MNF116]), 82%/37% (p=0.01); cytokeratin 3 (CK3), 74%/25% (p=0.009); cytokeratin 19 (CK19), 46%/25% (p=0.19); vimentin, 56%/53% (p=0.48); delta N p63α, 54%/0% (p<0.001); and ABCG2, 5%/0% (p=0.1). Flow cytometry showed a higher percentage of small cells, a higher percentage of MNF116+ cells, and stronger expression of progenitor-associated markers in superficial than in full-thickness explant cultures. For superficial limbal explant cultures, analysis of the expression profiles for various mRNAs at the end of 21 days of culture showed high levels of expression of the mRNAs encoding CK3, vimentin, and CK19. The expression of mRNA of delta N p63α and ABCG2 was weaker. Cultures obtained from full-thickness limbal explants featured no expression of mRNA of CK19, delta N p63α, and ABCG2, whereas mRNAs encoding CK3 and vimentin were detected. Human corneal stromal explants cultured with the same medium featured late cell growth, large mean cell area (2,529 µm(2)), no expression of cytokeratins, delta N p63α, and ABCG2, and high expression of vimentin.
  
  
  CONCLUSIONS
  Superficial limbal explants appear to be superior to full-thickness limbal explants for growing human limbal epithelial cells. Preparation of explants using surgical facilities (i.e., operating microscope and microsurgical blades) led to a dramatic increase in the percentage of successful cultures, higher epithelial cell growth, decreased fibroblast contamination, and better preservation of limbal epithelial progenitors.
authors.affiliation: Institut de la Vision, UPMC Univ Paris 06, UMR_S 968 / INSERM, U968 / CHNO des XV-XX / CNRS, UMR_7210, Paris, France. benallaouadjida@yahoo.fr
authors.forename: D
authors.initials: D
authors.lastname: Ghoubay-Benallaoua
authors.forename: E
authors.initials: E
authors.lastname: Basli
authors.forename: P
authors.initials: P
authors.lastname: Goldschmidt
authors.forename: F
authors.initials: F
authors.lastname: Pecha
authors.forename: C
authors.initials: C
authors.lastname: Chaumeil
authors.forename: L
authors.initials: L
authors.lastname: Laroche
authors.forename: V
authors.initials: V
authors.lastname: Borderie
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000737:chemistry; D003315:Cornea / Q000378:metabolism / Q000473:pathology*; D016039:Corneal Transplantation; D004847:Epithelial Cells / Q000166:cytology*; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007700:Kinetics; D016850:Limbus Corneae / Q000378:metabolism / Q000473:pathology*; D018613:Microscopy, Confocal / Q000379:methods; D008875:Middle Aged; D010641:Phenotype; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology
chemical_list: D002772:Cholera Toxin