Epithelial Cell Media
F12 + 5% FBS
Insulin 10ng/ml, EGF 10µg/ml, Hydrocortisone 10µg/ml
(Pen, Strp, Fung)
Fibroblast Media
DMEM+ 10% Calf Serum
(Pen, Strp, Fung)
- Keep the breast tissue on ice until dissociation. (Can hold for several hours after harvest)
- 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. - Incubate the tubes on a rotator at 37oC overnight. (or until the large tissue fragments are dissociated).
- Remove the tubes from incubator and then leave to stand for 2-5minutes to allow organoids to settle.
- Carefully decant the supernatant to a fresh tube. This contains the single-cell stromal cells.
- Wash the tube containing the organoids fraction with PBS+5%FBS and centrifuge all tubes (from step 6&7) for 5 min.
- Wash all tubes with 10mls of PBS+5%FBS and spin tubes 3 more times.
- Either plate cells/organoids in appropriate media or freeze in correct freeze media.
Collagenase Solution
3mg/ml Collagenase
125units/ml Hyaluronidase in above media
Pen/Srp/Fung