Xenobiotic- and Serum-Free Culture Protocol for Autologous Cultivated Oral Mucosal Epithelial Cells on Therapeutic Contact Lenses

Abstract

Purpose: : Cultured autologous oral mucosal epithelial cells (OMECs) have proven useful in the treatment of ocular surface disorders. However, the ideal culture period prior to transplantation is unknown. This study is the first to investigate the potential of expanding OMECs in a serum-free media using therapeutic contact lenses as a substrate and carrier.

Methods: : Porcine OMECs were seeded on laminin-coated lotrafilcon A therapeutic contact lenses (AirOptix Night&Day, CIBA VISION, Atlanta, GA, USA) with the density of 100000 cells/lens, and cultured in a defined serum-free medium (CnT-24, CELLnTEC, Bern, Switzerland). Cultures were monitored by phase contrast microscopy. Cellular morphology and phenotype were evaluated by confocal immunofluorescence microscopy using rhodamine-phalloidin staining of F-actin and antibodies against putative stem cell markers (ABCG2 and p63), and cell viability was measured using propidium iodide (PI) and Hoechst 33342 dual staining.

Results: : Porcine OMECs attached well to the contact lenses and exhibited confluent cobblestone monolayer morphology and loosely attached cells on the top of the monolayer after both 3 and 6 days. Basal layer cells showed an undifferentiated phenotype following 3 and 6 days of culture. Expression of ABCG2 in the basal cell layer was 6.3 ± 1.0% and 4.8 ± 1.8% after 3 and 6 days culture, respectively (p=0.053). No significant difference in expression of p63 was observed after 3 days culture (79.4 ± 14.8%) compared with 6 days culture (60.3 ± 18.9%)(p=0.078). The basal layer viability of cultured OMECs was 99.3 ± 0.5% and 82.8 ± 2.% after 3 and 6 days culture, respectively (p<0.05).

Conclusions: : These findings demonstrate that a xenobiotic- and serum-free culture system using cultured OMECs on therapeutic contact lenses is a promising platform for ocular surface reconstruction. Further studies are warranted to facilitate proliferation and stratification, and enhance the long-term viability of OMECs.

Keywords: cornea: epithelium • contact lens • cell adhesions/cell junctions