Begin by dissociating the tissue. Use two pairs of forceps to remove the adipose tissue from the mammary tissue. Ensure that the remaining mammary tissue weighs approximately one gram.
Rinse the mammary tissue in five milliliters of 75%ethanol solution for five seconds. Then, wash with 20 milliliters of washing solution two times for five minutes each. Then, use two surgical blades to slice the mammary tissue into smaller fragments.
Continuously for 15 minutes to obtain a tissue homogenate, transfer the shredded tissue to a 50 milliliter centrifuge tube. Add 10 milliliters of dispase and collagenase solution, three milliliters of 0.25%trypsin, and seven milliliters of PBS for a total of 20 milliliters of digestion solution. Place the tube in a water bath at 37 degrees Celsius and incubate for 1 1/2 hours, shaking the tube every 20 minutes.
To stop the digestion process, add 20 milliliters of neutralizing solution. Then, pipette the contents approximately 15 times to ensure thorough mixing. Filter the mixture through a 100 micron mesh filter.
After that, centrifuge at 156G for five minutes. Then, remove the supernatant. Repeat incubation of the pellet with 20 milliliters of neutralizing solution, then mix by pipetting.
Centrifuge again for five minutes. Carefully remove the supernatant and resuspend the cell pellet with 10 milliliters of initial culture medium. Plate the cell suspension in a 100 millimeter cell culture dish.
Cultivate the cells in a 5%carbon dioxide incubator at 37 degrees Celsius. Replace the original culture medium with fresh epithelial cell medium every third day. Check the cells and refresh the medium every two days.
Using this protocol, human mammary epithelial cells were isolated from mammary tissue. Post isolation analysis showed that cells treated with ROCK inhibitor Y-27632 exhibited pronounced growth compared to the control group. CCK-8 assay revealed that cells in the medium containing Y-27632 proliferated at a significantly higher rate than those in the control medium.
Immunofluorescence assays confirmed that the majority of cells expressed mammary epithelial cell markers, CK7 and GATA3, with negligible presence of other cell types in subsequent passages. Further, qRT-PCR studies showed consistent expression levels of CK7 and GATA3 over several passages indicating consistent phenotype or differentiation capacity.
