Abstract

SO.04.09

Phenotypic characterization of cultured human oral mucosal epithelial sheets for the purpose of ocular surface reconstruction

Kruse F. E.¹, Nkenke E.², Blazejewska E.¹, Hofmann-Rummelt C.¹, Schlötzer-Schrehardt U.^1
1Department of Ophthalmology, ²Department of Oral and Maxillofacial Surgery, University of Erlangen-Nürnberg, Erlangen,

Objective: Transplantation of autologous oral mucosal epithelial stem cells may be an alternative to allograft transplantation in patients with total bilateral limbal stem cell deficiency (Nakamura et al. 2003). The aim of this study was to characterize the phenotype of expanded oral mucosal epithelial cells under various culture conditions and to evaluate their potential for clinical applications.
Methods: Buccal mucosa biopsies were obtained from 10 patients undergoing dental surgery and cultured as explants or single cell suspensions on fibrin gels or amniotic membrane for 2 to 3 weeks in the presence of a 3T3 feeder layer in DMEM/F12. Epithelial stem cell populations were analyzed by colony-forming assays on 3T3 fibroblasts. The epithelial phenotype was evaluated by light and electron microscopy and immunohistochemistry using antibodies to cornea-specific differentiation (K3/K12) and stem cell markers (ABCG2, p63, integrin b1).
Results: Oral mucosal epithelial cell suspensions or explants cultivated both on fibrin gels or amniotic membrane produced confluent epithelial cell sheets with 3 to 5 layers of stratification resembling corneal epithelium. Clonal analysis produced stable large colonies forming holoclones from 0.2-0.3% of seeded cells, which could be subcultured on feeder cells for more than 20 passages and subcultured on carrier substrates as confluent monolayers. By electron microscopy, the epithelial cells were well-differentiated with formation of prominent tonofilaments, desmososmes, and hemidesmosomes in the primary cell sheets, but appeared less differentiated in the subcultured sheets. Immunohistochemistry identified the presence of cornea-specific marker K3/K12, and indicated the retention of progenitor cells positive for ABCG2, p63, and integrin b1 in cultivated cell sheets.
Conclusions: Oral mucosal epithelial sheets with phenotypic characteristics of corneal epithelium can be expanded on transplantable carriers such as fibrin gels or amniotic membrane by various culture techniques. The findings support the feasibility of a novel autologous tissue engineering approach for ocular surface reconstruction in patients with total bilateral limbal stem cell deficiency.

Copyright: Porstmann Kongresse GmbH. All rights reserved. Impressum, Disclaimer.