2.5 | LF Cell Culture
The LF specimens obtained from patients during surgery were washed 3 times with phosphate-buffered saline (PBS, Gibco), minced into into small fragments of approximately 0.5mm3 and digested using 0.2% type I collagenase (Gibco) at 37℃ for 1.5h. Subsequently, the digested pieces were washed with Dulbecco’s modified Eagle’s medium (DMEM, Gibco) and cultured with DMEM supplemented with 10% fetal bovine serum (Sigma), 100 U/ml penicillin and 100 pg/ml streptomycin (Sigma) in a 5% CO2 humidified incubator at 37℃. The cell-culture medium was changed every two to three days. The second passage of cells was used for the subsequent experiments.
The expression levels of collagen I and Ⅲ were detected by immunostaining and observed under a confocal microscope (XTL3230-DIC, Shanghai, China) to identify the cell type. The second passage of LF cells was cultured and stimulated with with different concentrations of recombinant human WISP-1 ranging from 0 to 150ng/ml for 24 h. For Hedgehog signaling inhibition experiments, 106 cells were seeded in each well of 6-well plates. Then, the indicated dose (5μM) of inhibitor (cyclopamine, Sigma, USA) without cytotoxicity were added in the culture medium for 24 h. Finally, cells were collected for subsequent experiments.