Effects of Media Composition on the Phenotype of Oral Mucosal Epithelial Cell Sheet Cultured for Corneal Epithelium Reconstruction

Abstract

Purpose : Safety of cellular products remains the most important criterion for human applications. FDA encourages the use of xeno-free cell culture conditions to minimize the risk of transmitting disease or causing human immune reactions. The aim of this study was to examine the effect of xeno-free culture medium and to examine the impact of different feeder cells on cell growth and cell morphology.

Methods : A biopsy of rabbit buccal tissue was used to isolate and seed oral mucosal epithelial cells (OMECS) on a temperature-responsive cell culture surface. OMECS were expanded in coculture with MMC immortalized NIH3T3 mouse embryonic fibroblasts (MEFs) or with human foreskin fibroblasts (HuFFs). Standard media (STDM) that include animal or bacterial derived products, such as fetal bovine serum and cholera toxin, was used in comparison to xeno-free media (XFM) containing only GMP-compatible cell products. Western Blot and morphologic analysis were used to examine the impact of feeder cells and media on cell sheet growth and phenotype.

Results : Both feeder cells MEFs and HuFFs similarly supported cell growth of rabbit OMECS. Rabbit OMECS grew faster in STDM than in XFM. The microscopic examination showed that cell morphology was more homogeneous in XFM than in STDM conditions. All the cell sheets showed similar expression of deltaNp63 and Pax-6. However, cell sheets grown on XFM showed a lower expression of Occludin, E-cadherin and Connexin43, as compared to the cell sheet grown with STDM.

Conclusions : These findings demonstrated that HuFFs successfully supported oral mucosal epithelial cell sheet growth and also demonstrated that medium composition directly regulates junctional complexes and might be responsible for cell sheet phenotype and epithelial integrity.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.