4.4. Separation and Culturing of the Primary Intestinal Epithelial Cells

After an adaptation period of 3 d, the young mice were executed, after which the intestinal tissue was excised and washed three times with ice-cold PBS buffer. Then, the tissue was cut into small pieces of approximately 1 mm³ and immersed in a DMEM/F12 medium containing a penicillin-streptomycin solution (100×) for 1 h. The supernatant was collected and mixed with collagenase XI (450 U/mL) and neutral protease (0.2 mg/mL) for digestion at 37 °C for 20 min, after which the sample was centrifuged (1500 g, 15 min), and the precipitate was saved. The cells were seeded into a 6-well plate, which was pre-coated with collagenase I (450 U/mL). After most of the cells were attached to the wells, the small intestinal epithelial cell colonies were screened, and the fibroblasts were discarded by washing and digesting several times. Finally, basically all the intestinal epithelial cells appeared in the microscopic field of vision, which could be utilized for the further experiments.