Methods: Epithelial cells and mesenchymal cells were prepared from the mandibular incisors of 10-day-old rats and the former cells were inoculated to the collagen solution within a φ 4.5mm plastic cylinder. After the collagen gel had been polymerized, dental epithelial cells were layered on the gel containing dispersed mesenchymal cells, thus combining the three-dimensional culture of dental mesenchymal cells with layered culture of dental epithelial cells, which we designated the 3D & layered culture (TDL culture). One day after inoculation, the TDL gel was removed from the cylinder and floated in the medium (TDL floating culture). The TDL gels were then fixed, embedded, and processed for routine histological examinations. The cells inoculated to collagen-coated 96well-plate for the mono-layered cultures were used as control.
Results: During TDL culture, the gel shrank to 1/3-1/5 in size depending on the cell density. Undistorted shrinkage of the gel and cell layers was attained when the proportion of epithelial/mesenchymal cell density was 1:2. Proliferation, differentiation, and morphology of both types of cells differed depending on the culture medium used, but cells in TDL culture generally showed better morphology compared to that in mono-layered culture. Further, matrix like structure was deposited at the interface between the layers of epithelial and mesenchymal cells. Processes of mesenchymal cells extended vertically to the epithelial cell layer in the upper marginal region of the gel but much less in the deeper layers away from the interface.
Conclusion: Our TDL culture is suitable for both epithelial and mesenchymal cells prepared from rat incisors, and useful for in vitro analyses of epithelial-mesenchymal interactions.