Primary Culture Of Mouse Embryonic Palatal Epithelial Cells In Vitro And Observation Of The Primary Cilia

Objective: Through the cultivation of mouse embryonic palatal epithelial cells in vitro, to establish a reliable method for primary culture of embryonic palatal epithelial cells in mouse; Separation and culture of mouse embryonic palatal mesenchymal cells in vitro to observe the expression of primary cilia within. Lay the foundation for subsequent study.Methods: The embryonic palatal shelves were dissected from gestation day(GD) 13.5 mouse fetus under a stereomicroscope, then the embryonic palatal shelves were processed with 0.4% dispase II at 4℃overnight, epithelial and mesenchymal tissue were separated by mechanical isolation on the next day. 1The epithelial tissue were dissociated with 0.05% trypsin, and then seeded into plastics culture plate with Medium 171 medium(supplemented with 5% fetal bovine serum and 5ng/ml EGF). Cell morphology and growth were observed, and identified by immunocytochemical staining with anti-cytokeratin and anti-vimentin monoclonal antibody. 2The mesenchymal tissue were dissociated with 0.25% trypsin+0.02% EDTA into single cell suspension,and then seeded into plastics culture plate with DMED/F12 medium(supplemented with 10% fetal bovine serum). Cell morphology and growth were observed, and identified by immunocytochemical staining with anti-cytokeratin and anti-vimentin monoclonal antibody. Immunofluorescence staining of αα-tubulin and γ-tubulin were performed to visualize primary cilia.Results: Highly purified embryonic palatal epithelial and mesenchymal cells in mouse can be obtained through using Dispase II and trypsin digestion in combination of the mechanical separation. Under microscope, 1the mouse embryonic palatal epithelial cells showed typical “cobble stone-like” morphology when reached confluency. Immunocytochemistry staining results showed the P-CK positive cells was 94% in cultured mouse embryonic palatal epithelial cells. 2The mouse embryonic palatal mesenchymal cells were spindle shaped and had a typical fibroblast like morphology.Immunocytochemistry staining results showed the vimentin positive cells was around 97% in cultured mouse embryonic palatal mesenchymal cells. Primary cilia can be identified by searching for γ-tubulin positive basal bodies proximate to αα-tubulin positive axonemes in mouse embryonic palatal mesenchymal cells.Conclusion: 1High purity mouse embryonic palatal epithelial and mesenchymal cells can be obtained by mechanical separation in combination of enzyme digestion method.2The mouse embryonic palatal mesenchymal cells have the primary cilia.