Human epithelial cell cultures from superficial limbal explants

[摘要] Purpose: To study the kinetics of growthand the phenotype of cells cultured from human limbal explants in acholera toxin-free medium with no feeder cell layer. Methods: Human organ-cultured corneaswere used to prepare limbal explants (full-thickness and superficiallimbal explants) and corneal stromal explants. Cell growth kinetics andphenotypes were assessed by cultivating explants in cholera toxin-freeGreen medium. Epithelial and progenitor cell markers were assessed byimmunocytochemistry, flow cytometry, and Reverse Transcription andPolymerase Chain Reaction (RT-PCR). Results: The successful epithelial cellgrowth rates from full thickness limbal explant and superficial limbalexplant tissues were 41 and 86%, respectively (p=0.0001). The mean cellarea and the percentage of small cells in superficial andfull-thickness explant cultures were, respectively, 317 µm2and 429 µm2, and 8.9% and 1.7% (p<0.001). The percentageof positive cells in superficial and full-thickness limbal explantcultures as assessed by immunocytochemistry were the following: broadspectrum 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). Flowcytometry showed a higher percentage of small cells, a higherpercentage of MNF116+ cells, and stronger expression ofprogenitor-associated markers in superficial than in full-thicknessexplant cultures. For superficial limbal explant cultures, analysis ofthe expression profiles for various mRNAs at the end of 21 days ofculture showed high levels of expression of the mRNAs encoding CK3,vimentin,and CK19. The expression of mRNA of delta N p63αand ABCG2 was weaker. Cultures obtained from full-thicknesslimbal explants featured no expression of mRNA of CK19, deltaNp63α, and ABCG2, whereas mRNAs encoding CK3 andvimentin were detected. Human corneal stromal explants cultured withthe same medium featured late cell growth, large mean cell area (2,529µm2), no expression of cytokeratins, delta N p63α, andABCG2, and high expression of vimentin. Conclusions: Superficial limbal explantsappear to be superior to full-thickness limbal explants for growinghuman limbal epithelial cells. Preparation of explants using surgicalfacilities (i.e., operating microscope and microsurgical blades) led toa dramatic increase in the percentage of successful cultures, higherepithelial cell growth, decreased fibroblast contamination, and betterpreservation of limbal epithelial progenitors.

[学科分类] 生物化学/生物物理