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So-Hyang Chung, Sohee Jeon, Seong Hyun Choi, Jose Wolosin, Choun-Ki Joo; Regeneration of corneal epithelium with conjunctival epithelial equivalents generated in serum and feeder cell-free medium. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5160.
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An alternative autologous tissue for ocular surface reconstruction is a potential treatment for the patients with bilateral limbal stem cell deficiency. For the purpose of regenerative procedures in patients, it will be desirable to eliminate the involvement of xenogeneic components, such as non-human sera and feeder cell. In the present study, we examined the behavior and phenotypic features of cultured conjunctival epithelial sheets generated in serum- and 3T3- free culture conditions when transplanted into the de-epithelialized limbal corneal surface.
Epithelial cells from normal conjunctiva obtained by neutral protease digestion were expanded by culture in a serum-free low calcium medium, and set in air-liquid interface culture for 14 days. Resulting multi-layered epithelial sheets were grafted onto rabbit ocular surfaces made epithelial-free by alkali treatment. Pre-grafted and post-grafted epithelia were analyzed by electron microscopy and immunohistochemistry.
At graft time the cultured epithelial sheet consisted of 6-8 layers of properly stratified epithelium that displayed a CK19+/MUC5AC+/ CK3 -/ CK12- phenotype, consistent with the conjunctival epithelial lineage. Two weeks after xeno-grafting the in vivo epithelium consisted of 5-6 well compacted layers expressing an established anti-human nuclei antibody, the precursor cell-related protein p63, the proliferation marker Ki67, desmosomes, hemidesmosomes and its integrin (β4), and the corneal specific cytokeratins CK3, and CK12. Conjunctival Goblet cell mucin (MUC5AC) was not visible.
Our results suggest that conjunctival epithelial sheets generated in serum- and 3T3- free culture conditions can acquire the corneal epithelial phenotype when transferred to the in vivo corneal stromal environment.
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