Medium-term Culture of Normal Human Oral Mucosa: A Novel Three-dimensional Model to Study the Effectiveness of Drugs Administration

Abstract

Tissue-engineered oral mucosal equivalents have been developed for in vitro studies for a few years now. However, the usefulness of currently available models is still limited by many factors, mainly the lack of a physiological extracellular matrix (ECM) and the use of cell populations that do not reflect the properly differentiated cytotypes of the mucosa of the oral cavity. For this reason, we have developed a novel three-dimensional culture model reflecting the normal architecture of the human oral mucosa, with the main aim of creating a better in vitro model where to test cellular responses to drugs administration. This novel 3D cell culture model (3D outgrowth) was set up using an artificial extracellular matrix (Matrigel TM ), allowing the interactions required for proper differentiation of the various citotypes which form the mucosal layer. Biopsies of human oral mucosa, in fragments of about 0.5 mm 3 , were placed onto 6.5mm Transwells, covered with Matrigel TM and grown in a specific culture medium. A gradual formation of an architectural structure similar to that of the in vivo oral mucosa was observed. Transmission electron and confocal microscopy were employed to characterize the newly developed model: the cell components (keratinocytes and fibroblasts) differentiated properly within the outgrowth and reconstituted, in vitro, the physiological structure of the human oral mucosa, including a stratified non-keratinized squamous layer composed of four different layers, a proper basal membrane and a lamina propria where fibroblasts produce ECM. Moreover, keratinocytes expressed CK5, CK13, CK19 and E-cadherin, whereas fibroblasts expressed collagen type I and IV, laminin and fibronectin. 3D outgrowths could be considered a valid alternative to animal models, and provide useful information for researchers interested in studying the responses of the human oral mucosa to locally delivered drugs or other exogenous treatments.

Characterization of Ex Vivo Expanded Oral Mucosal Epithelium Cells on Acellular Porcine Corneal Stroma for Ocular Surface Reconstruction

“…COMECs that are absent of xenobiotic feeder cells such as murine 3T3 feeder layers have the advantage of avoiding ethical and pathogen transmissions and providing an unlimited cell source as an alternative therapy for reconstruction of ocular surfaces in patients with bilateral LSCD. For this reason, in recent years, COMECs procedures that are feeder cell-free have dominated in terms of studies, including temperature-responsive cell culture wells [22, 49], fibrin-coated culture inserts [18], laminin-coated CLs [26], cultures coated with Matrigel [50], and HAM [10, 27–35]. Nowadays, more and more researches developed techniques for the COMECS protocol with the use of xenobiotic feeder-free as well as serum-free culture systems [51, 52].…”

Functional characterization of a novel 3D model of the epithelial-mesenchymal trophic unit

“…At Days 10, 20 and 30, cultures were fixed, post-fixed and dehydrated before embedding in Epon resin, as previously described (19). Ultrathin (50nm) sections were then cut and placed on Cu/Rh grids.…”

Medium-Term Culture of Primary Oral Squamous Cell Carcinoma in a Three- Dimensional Model: Effects on Cell Survival Following Topical 5-Fluororacile Delivery by Drug-Loaded Matrix Tablets

“…The method employed to obtain 3D human oral outgrowths from normal human oral mucosa has already been described [29]. The 3D OSCC outgrowths were obtained similarly.…”

“…In the present work we have used a novel 3D in vitro model that enabled us to perform mid-to longterm cultures of both normal and malignant oral mucosa [29]. Using this model could lead to an increased understanding of the features of OSCC progression and to the discovery of new potential therapeutic strategies.…”