Objective: To further investigate the mechanisms of hair cell generation or regeneration, the primary culture systems of cochlear sensory epithelial cell (CSEC) of rats were established.
Method: Cochlear sensory epithelial (containing the organ of Corti) of postnatal day 1 (P1) rats was isolated by mechanical dissociation. The explants were grown on sterilized disposable plastic dishes,cultured in Dulbecco modified Eagle medium(DMEM), and observed daily by inverted microscopy. The culture medium was changed twice a week. The pure CSEC was harvested by the limiting dilution method. CSEC was identified by immunocytochemical method with cytokeratin 18, vimentin, Brn3. a, Calretinin, BrdU and ultrastructural examination with transmission electron microscopy. The markers of epithelial cell and the markers of hair cell were used to identify the origin and character of CSEC. CSEC mitotic division was detected by BrdU staining of nuclear DNA.
Result: The fresh explants were light yellow. The morphology of CSEC couldn't be seen clearly under the inverted microscope because of the complex structures of cochlear sensory epithelial. CSEC grew out of the explants usually at 2nd culture day. Fibroblast like cells (FLC) around the CSEC grew faster than CSEC, but could easily be excluded. Pure CSEC may grow into monolayer with 'cobblestone-like' appearance and show a large, flat, polygonal epithelial morphotype with big, round nuclei. Some cells showed 'Dome' formation, probably due to fluid collection underneath the cell monolayer. The culture CSEC coexpressed cytokeratin 18 and vimentin has rich microvilli and complex tight junction,which indicated the epithelial origination of CSEC. Coexpressed of the Brn3. a and Calretinin of the hair cell characteristic markers suggested the culture cell may represent rat progenitor hair cell. BrdU staining showed CSEC produced new hair cell-like cell (progenitor hair cell) by the mitotic division.
Conclusion: The primary culture systems of cochlear sensory epithelial cell of rats were successfully established in vitro. CSEC coexpressed the characteristic markers of the immature hair cell,which identified the culture cell may represent progenitor cell of rats hair cell. It may be a suitable model for in-depth investigation the molecular mechanisms of hair cell generation or regeneration.