10X Collagenase Solution: 15mg/ml collagenase, 1250 units/ml hyaluronidase, and 1X Pen/Strep/Fung in epithelial cell media, stored at -20°C. For each 1-2gms of tissue, use 10mls of a 1:5 diluted solution for a final working solution of 3mg/ml collagenase, 250 units/ml hyaluronidase, and 1X Pen/Strep/Fung in epithelial cell media. The longer you wish to digest the tissues, the more dilute the solution can be; thus, a 1X solution will work, it just requires longer dissociation.
PROCEDURE
Human Breast Organoid separation
1 Sterilize instruments using a bead sterilizer or bunsen burner.
2 Bring the tissue into a clean, sterile tissue culture hood and remove yellow adipose tissue with operating scissors.
3 Mince the sample until the pieces of tissue are approximately 3-5 mm3 with sterile razor blades.
4 Transfer 1-2 grams of tissue to a 15 ml conical tube filled with 10 ml of working collagenase solution.
5 Incubate the tubes on a rotator at 37°C until the large tissue fragments are dissociated (typically 8-12 hours, but can go overnight).
6 Remove the tubes from the incubator and let them stand for 2-5 minutes to allow organoids to settle. These large clusters of tissue represent the epithelial portion, and are thus composed of luminal epithelial and myoepithelial cells.
7 Decant the supernatant to a fresh tube.
PAUSE POINT – This supernatant contains single-cell stromal cells, and can therefore be grown in fibroblast media to expand, can be frozen back or simply discarded.
8 Wash the organoid-containing tube with PBS + 5% FBS and centrifuge (300xg on a table top centrifuge at 4°C) for 5 minutes.
9 Repeat step 8 three more times with 10 mls of PBS + 5% FBS (if stromal cells are to be plated, they should be washed in a similar fashion).
PAUSE POINT – At this point, each conical tube of organoids can be frozen back as single aliquots in cryovials by resuspending organoids in 900 ml of epithelial media and 100 ml of DMSO, and storing at -80°C.
10 At this point, cells can be plated to generate primary epithelial cells or used for generating normal structures in the mammary fat pad as discussed below. If plating the organoids, resuspend in 10% of the volume of your desired culture plate in organoid plating media, plated drip wise, wait 1-2 minutes to allow the organoids to sit, and slowly add the remaining 90% of media. Like all mammalian cells in this protocol, incubate cells in a humidified 37°C tissue culture incubator with 5% CO2, feeding with the appropriate medium (pre-warmed) every other day after aspirating the old medium off. Leave cells in the plating media until the fibroblasts are no longer present, but if the cells approach confluence, the cells should be split (this will accelerate fibroblast loss). One will notice long, spindle type cells versus the smaller cobblestone epithelial cells. Eventually these long fibroblasts will senescence, enlarge, and die and generally take about one to two weeks. From then on, the epithelial cells should be cultured in MEGM. For more detailed information on culturing primary HMECs please see Martha Stampfer’s excellent review of the HMEC culture system at http://www.lbl.gov/~mrgs/.
11 To generate immortal fibroblasts from primary fibroblast cultures, infect a population with a retroviral construct generated to encode for the catalytic subunit of human telomerase (hTERT)8, and subsequently select the cells using a mammalian selection marker or by cell sorting depending upon the viral construct backbone.
