Stability of Cultured Dental Follicle Cells

Objectives: Because the dental follicle is required for tooth eruption, establishment of dental follicle cell (DFC) lines is needed for experimentation. In long-term cultures, do the cells lose contact inhibition of growth (immortalization) and/or differentiate into another cell type such as an osteoblast or osteocyte? To analyze the stability of the DFC cultures, different passages were analyzed by flow cytometry and von Kossa staining to measure contact inhibition and differentiation, respectively. Methods: Rat DFC were cultured in MEM containing 10 % newborn calf serum and 1 mM sodium pyruvate. Cells were passed at confluency until the desired passage. Approximately 3 x105 cells from each of the passages 6, 9, 12 were cultured per T-flask in 20 ml medium and collected from days 2-14 for cell cycle analysis using flow cytometry. To screen for calcium deposition, cells were grown on coverslips and subjected to von Kossa staining. Results: Within a given passage, 30-40 % of the cells were on a dividing track (S, G2, M) during the first two days in culture. After 4-5 days, the number of such cells declined to only 7-15 % of the total. At confluency, only 3 % and 7 % of the cells were in the dividing mode in passages 6 and 9 respectively, but this increased to 15 % in passage 12. The von Kossa staining showed no calcium deposition for all passages tested. Conclusion: The DFC are remarkably stable through passage 9 in terms of contact inhibition of growth. By passage 12, perhaps 15 % of the cells have lost contact inhibition. Thus, it is recommended that DFC of passage 9 or less be used for in vitro experimentation. No indication of cell differentiation into bone cells was detected for any passage. Supported by grant DE08911 from NIDCR to G.E.W.