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.
125units/ml Hyaluronidase in above media
Charlotte Kuperwasser, PhD
Associate Professor of Developmental, Molecular & Chemical Biology
Tufts University School of Medicine
Molecular Oncology Research Institute (MORI)
Tufts Medical Center
Boston, MA 02111