Isolation of HMECs from Redcution Mammoplasty Tissue

DMEM+ 10% Calf Serum

(Pen, Strp, Fung)

1. Keep the breast tissue on ice until dissociation.  (Can hold for several hours after harvest)
2. Using sterile razor blades, chop breast tissue into 3-5mm pieces.
   Transfer 1-2gm of breast tissue to a 15ml conical (polypropylene tube) containing 10ml of collagenase solution.
3. Incubate the tubes on a rotator at 37^oC overnight. (or until the large tissue fragments are dissociated).
4. Remove the tubes from incubator and then leave to stand for 2-5minutes  to allow organoids to settle.
5. Carefully decant the supernatant to a fresh tube. This contains the single-cell stromal cells.
6. Wash the tube containing the organoids fraction with PBS+5%FBS and centrifuge all tubes (from step 6&7) for 5 min.
7. Wash all tubes with 10mls of PBS+5%FBS and spin tubes 3 more times.
8. Either plate cells/organoids in appropriate media or freeze in correct freeze media.