Store at 4℃ away from light for 1 month.

1. Culture of primary breast epithelial cells

The human breast tissue was digested by digestive enzymes, filtered by a 100-micron screen, counted, and inoculated in a suitable culture vessel with NIH-3T3 cells irradiated by gamma rays. The primary cells were not moved for 2-3 days after the initial inoculation (conducive to cell adhesion). During the culture process, if the medium color turned yellow but the cells were not overgrown, the half solution could be changed. When NIH-3T3 cells are not overgrown but insufficient, NIH-3T3 cells irradiated by gamma rays are properly supplemented (generally half of the initial number).

2. Passage of primary mammary epithelial cells

The cells were observed to form clones under microscope and could be passed through when the confluence reached 80-90%. The cells were removed from the incubator, discarded the old culture medium, washed with 0.05% pancreatic enzyme for 30 seconds and sucked up, then added an appropriate amount of TrypLE™ Express enzyme (Gibco#12605010), incubated at 37℃ for 10-20 min, and gently tapped the side of the culture bottle/plate to observe the cell digestion under a microscope. After digestion, the cells became round and began to fall off, the digestion was terminated with DMEM medium containing 10% fetal bovine serum, and the cells were transferred to a centrifuge tube, 300g, and centrifuged for 5 min. The supernatant was discarded, the cells were re-suspended with 1-2 mL of this cell medium product, the live cells were counted, and the cell suspension was inoculated in the culture bottle/plate according to the appropriate cell density. After that, NIH-3T3 cells irradiated by gamma rays were added, the culture plate/hole was supplemented with mammary primary epithelial cell medium to the required volume, gently shaken and mixed, and the surface was disinfected and cultured in an incubator at 37 ℃ and 5% CO2. Do the same for other steps.