Cultivation of Human Oral Mucosal Explants on Contact Lenses

Abstract

ABSTRACTPurpose/Aim: Autologous cultivated oral mucosal (OM) epithelial transplantation has been successfully used as corneal epithelial replacement in bilateral limbal stem cell deficiency. Recently, lotrafilcon A contact lens (CL) surface was described as a suitable carrier for cultured stem cells in corneal epithelial transplantation. Our aim was to establish explant cultures from human OM on CL carriers that are free of animal-derived materials and feeder cells.Materials and methods: Human cadaveric 2 mm OM explants were sutured onto CL surfaces and cultivated with fetal calf serum (FCS) or human serum (HS) supplemented culture medium without feeder cells. Confluent cultures were harvested and evaluated morphologically with hematoxylin and eosin stain and with immunofluorescence microscopy for the presence of p63, vimentin and cytokeratins (CK) 3, 4, 13 and 14.Results: Confluent cell sheets covering the whole CL surface were produced from OM explants after 2 weeks of culture with HS and after 3 weeks with FCS. A basal layer consisted of small, vimentin, p63 and CK14 positive putative stem/progenitor cells, which were present in the whole cell sheet. Large, CK3, CK4 and CK13 positive, differentiated cells appeared to spread above this confluent layer.Conclusions: We have established an animal-free culture system from human OM explants on CL surface. The cultured OM sheets contain large numbers of putative stem cells including limbal-like CK3 and CK14 positive cells. This method can be adapted to good manufacturing practice (GMP) conditions and has, therefore, great potential for clinical use.