Separation of cochlear basilar membrane epithelial cells by enzymatic digestion with thermolysin and type I collagenase
MI Wenjuan
SONG Yongli
TIAN Keyong
CHEN Fuquan
ZHA dingjun
QIU Jianhua
Abstract：Objective To establish a new method for isolating progenitor cells from cochlear basilar membrane epi-thelium by combinatorial enzymatic digestion. Methods Basilar membranes were separated from the cochlea of P1-3 neo-natal SD rats. The isolated basilar membrane sheets were treated by trypsin digestion (Group A), thermolysin digestion (Group B), type I collagenase digestion (Group C), or combined thermolysin and type I collagenase digestion (Group D). Cells isolated by enzymatic digestion were collected and cultured under suspension and adherent conditions. The number of otospheres, dominance by epithelial cells and proliferation and differentiation of progenitor cells were evaluated. Results Cells collected in all four groups gave rise to otospheres, with positive nestin and BrdU expression. Under the differentia-tion condition, cells from basilar membrane epithelium differentiated into hair cell-like cells. The epithelial origin was con-firmed by cytokeratin-18 and E-cadherin staining. Epithelial cells dominance was the greatest in Group D and significantly lower than other three groups in Group A. Conclusions Combinatorial enzymatic digestion with thermolysin and type I col-lagenase can significantly improve isolation of epithelial cells. This method provides a new tool for cochlear progenitor cells research.
Keywords：Hair cell progenitorsCell cultureEpithelial cellsEnzymatic digestion
Publication Date：2016-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：5( 255-259 )