An In Vitro Culture System for Long-Term Expansion of Epithelial and Mesenchymal Salivary Gland Cells: Role of TGF-1 in Salivary Gland Epithelial and Mesenchymal Differentiation
Figure 3
In vitro culture of Col1a1-GFP derived submandibular salivary gland cells. Mixed salivary gland epithelial and mesenchymal cells (passage 1, for 2 weeks) exhibited different growth pattern and morphology when cultured in N2 media versus DMEM plus serum medium. (a)–(c) Mixed salivary gland cells were cultured in N2 media. A majority of cells grown in N2 media were polyhedral-shaped and GFP-negative, representing salivary gland epithelial cells (a and b). Some GFP+ mesenchymal cells were also found in this culture condition (b and c). N2 media enhanced sphere formation containing both salivary gland epithelial and mesenchymal cells (indicated by arrowheads) (a and c). (d)–(f) DMEM plus 10% serum promoted the growth of salivary gland mesenchymal cells which were shown as spindle-shaped and GFP-positive cells. Small round and GFP-negative cells were also observed on top of the mesenchymal or stromal monolayer, indicating the existence of salivary gland epithelial cells (d and e). (g)–(m) Quantitative specific gene expression was analyzed to confirm the presence of salivary gland epithelium and mesenchyme in both N2 and DMEM media plus serum. The expression of salivary gland epithelial genes, Amylase-1 (g), Aquaporin-5 (Aqp-5) (h), Zonula occludens-1 (ZO-1) (i), and Occludin (j), were significantly upregulated in N2 media-cultured cells. The expression of salivary gland mesenchymal genes, Fgf-7 (k), Fgf-10 (l), and Collagen type I (m), significantly increased in cells cultured in DMEM plus serum medium. Relative expression was normalized to the expression of Gapdh which was used as the reference gene. Values were represented as mean ± SEM from three independent experiments (). Student’s -test was analyzed to compare between cells cultured in N2 and DMEM media plus serum, ** and *. Scale bars = 100 μm.
