IADR Abstract Archives
in Vitro Oral Mucosa: ScRNA-seq-Based Modelling of Epithelial Barrier.
Objectives: The barrier function of oral epithelia is essential for maintaining tissue health. Complex, multi-layered structure of oral mucosa with stratified epithelium and collagen-rich connective tissue presents limitations for traditional in vitro models. Three-dimensional (3D) tissue culture provides a physiologically relevant alternative but understanding of epithelial stratification and barrier function development in the in vitro models is still limited. Our goal model epithelium stratification across in vitro grown tissues using single cell RNA sequencing (scRNA-seq).
Methods: Buoyant epithelial culture devices were employed to establish 3D models of oral mucosa, featuring collagen-based hydrogels seeded with epithelial cells on the surface and maintained for 3-4 weeks at air-liquid interface for epithelial maturation. The tissues were dissociated, 10,000 cells were recovered for library preparation and sequenced using NovaSeq 6000. In vitro tissues derived from primary gingival or tonsil keratinocytes, and from oral cancer cell line (VU40T), were compared. Lipopolysaccharide (LPS) was used to compromise epithelium integrity. Tissue structure was confirmed using H&E staining and epithelial integrity was assessed via transepithelial electrical resistance (TEER).
Results: In silico modelling was developed using single-cell RNA sequencing for keratinised, non-keratinised and cancer oral epithelium. All models showed clear epithelial maturation and spatial expression of differentiation markers, while terminal differentiation was only observed for keratinised epithelium. Immune function and cancer pathways were evident in tonsillar and cancer epithelia, respectively. Therefore, 3D in vitro epithelial tissues structurally and functionally represented their respective origins. The barrier integrity was observed to be reduced by LPS exposure and this was confirmed by decrease in tissue resistance.
Conclusions: These data increase understanding of epithelial stratification and barrier development in 3D in vitro models and confirm their physiological relevance. The obtained computational model forms a point of reference in future studies involving external challenges, genetic modifications and therapeutic approaches.
Methods: Buoyant epithelial culture devices were employed to establish 3D models of oral mucosa, featuring collagen-based hydrogels seeded with epithelial cells on the surface and maintained for 3-4 weeks at air-liquid interface for epithelial maturation. The tissues were dissociated, 10,000 cells were recovered for library preparation and sequenced using NovaSeq 6000. In vitro tissues derived from primary gingival or tonsil keratinocytes, and from oral cancer cell line (VU40T), were compared. Lipopolysaccharide (LPS) was used to compromise epithelium integrity. Tissue structure was confirmed using H&E staining and epithelial integrity was assessed via transepithelial electrical resistance (TEER).
Results: In silico modelling was developed using single-cell RNA sequencing for keratinised, non-keratinised and cancer oral epithelium. All models showed clear epithelial maturation and spatial expression of differentiation markers, while terminal differentiation was only observed for keratinised epithelium. Immune function and cancer pathways were evident in tonsillar and cancer epithelia, respectively. Therefore, 3D in vitro epithelial tissues structurally and functionally represented their respective origins. The barrier integrity was observed to be reduced by LPS exposure and this was confirmed by decrease in tissue resistance.
Conclusions: These data increase understanding of epithelial stratification and barrier development in 3D in vitro models and confirm their physiological relevance. The obtained computational model forms a point of reference in future studies involving external challenges, genetic modifications and therapeutic approaches.