Abstract
Transplantation of cultured oral mucosal epithelial cells (OMECs) is a promising treatment strategy for limbal stem cell deficiency. In order to improve the culture method, we investigated the effects of four culture media and tissue harvesting sites on explant attachment, growth, and phenotype of OMECs cultured from Sprague-Dawley rats. Neither choice of media or harvesting site impacted the ability of the explants to attach to the culture well. Dulbecco’s modified Eagle’s medium/Ham’s F12 (DMEM) and Roswell Park Memorial Institute 1640 medium (RPMI) supported the largest cellular outgrowth. Fold outgrowth was superior from LL explants compared to explants from the buccal mucosa (BM), HP, and transition zone of the lower lip (TZ) after six-day culture. Putative stem cell markers were detected in cultures grown in DMEM and RPMI. In DMEM, cells from TZ showed higher colony-forming efficiency than LL, BM, and HP. In contrast to RPMI, DMEM both expressed the putative stem cell marker Bmi-1 and yielded cell colonies. Our data suggest that OMECs from LL and TZ cultured in DMEM give rise to undifferentiated cells with high growth capacity, and hence are the most promising for treatment of limbal stem cell deficiency.
Highlights
Clinical results compared to the use of more differentiated equivalents
We found that oral mucosal epithelial cells (OMECs) from the lower lip and transition zone of the lower lip cultured in Dulbecco’s modified Eagle’s medium/Ham’s F12 (DMEM) give rise to undifferentiated cells with high growth capacity, and are the most promising for treatment of LSCD
OMECs were harvested from four sites in the oral cavity of rats and cultured in four different media (EpiLife, oral keratinocyte medium (OKM), DMEM, and Roswell Park Memorial Institute medium (RPMI)) and the explant attachment, cell outgrowth, proliferation, and phenotype of the cells were compared
Summary
Clinical results compared to the use of more differentiated equivalents. it may be clinically important to determine how the phenotype of cultured OMECs is influenced by the choice of tissue harvesting site and culture medium. The culture media investigated in this study were chosen for the following reasons: EpiLife is serum-free and has a low calcium-content, which is known to promote an undifferentiated phenotype[8, 9]; oral keratinocyte medium (OKM) with oral keratinocyte growth supplement, which includes pituitary extract, protects cells from H2O2-induced cell death, DNA fragmentation, and has a positive effect on cell viability[10]; Dulbecco’s modified Eagle’s medium/Ham’s F12 (DMEM) and Roswell Park Memorial Institute 1640 medium (RPMI) are among the most commonly used cell culture media. No studies have compared various harvesting sites for ex vivo expanded OMECs. As the phenotype, degree of keratinization, and morphology of the oral mucosa vary throughout the oral cavity[23, 24], we hypothesized that the harvesting site could affect the growth capacity and phenotype of ex vivo expanded OMECs. In the current study, the effects of harvesting site and culture medium on attachment, growth, and phenotype of cultured OMECs were investigated. We found that OMECs from the lower lip and transition zone of the lower lip cultured in DMEM give rise to undifferentiated cells with high growth capacity, and are the most promising for treatment of LSCD
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