Abstract: Objective: The odontogenic epithelial cells largely disappear after tooth eruption. It is proved that the non-odontogenic epithelial cells can be used as alternative seed cells of tooth regeneration. This study will characterizes the role of conditioned culture media on expansion of non-odontogenic epithelial cells in vitro for potential seed cells of further tissue engineering tooth. Method: The oral mucosa epithelial cells were harvested from 1day postnatal mouse and cultured to confluency in conditioned media supplemented with Y27632, then passaged and plated onto 3T3 feeder cells in conditioned media with Y27632 for continuous passage. Histology and immunohistochemistry were performed for biological characteristics analysis of cells. Results: Compared with the traditional ways of epithelial cell culture, the oral mucosa epithelial cells plated onto 3T3 feeder cells in conditioned media supplemented with Y27632 grew rapidly, which showed a cuboidal, polygonal or pavement-like shape and expressed CK14, SOX2 and LGR5. After continuous passage, the cell morphology, growth curve and cell markers have no obvious changes. Conclusion: The conditioned culture media supplemented with Y27632 have potential to effectively expand non-odontogenic epithelial cells in vitro, and maintain the characteristics of stem cells, which is expected for tissue engineering tooth as seed cells.