Enrichment and culture of primary mammary fibroblasts (PJ Keller)
Primary Mammary Fibro and Org culture ( Click to Download )
Starting from whole human mammary tissue
*based on the Nature Protocols Paper: Proia and Kuperwasser. Nat Protoc. 2006;1(1):206-14.
- Mince tissue into 3-5 mm pieces and digest overnight (12-16 hours) in a mixture of 1X collagenase and 1X hyaluronidase in organoid medium
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- Use approximately 2X volume of digestion mixture to tissue (ex. Add enough tissue to 10 ml of collagenase mixture in a 15 ml conical tube to fill to about 14-15 ml total volume
- Leave enough headspace so tissue can tumble freely
- Rotate tubes at 37C for digestion, seal tubes with parafilm to prevent leaking
- Allow organoid pieces (clusters of epithelial cells) to settle briefly; either let stand 1-2 minutes or spin at low speed (300 rpm) for 30 seconds to 1 min
- Crudely fractionate stromal cells from organoids: transfer the supernatant to a clean tube (leave some behind with the organoid pellet to ensure organoids are left behind). The supernatant fraction contains some organoids but also mainly single cells and stromal cells.
- Resuspend the organoids in PBS + 5% calf serum. Pellet the organoids and the enriched stromal cells (supernatant) at 1500 rpm 5’.
- Wash the stromal pellet and the epithelial organoid pellet 3 times with PBS containing 5% calf serum.
- Freeze back cells in media containing 10% serum and 10% DMSO or proceed with culturing.
Culture of primary fibroblasts and epithelial cells
Fibroblast cultures can be generated from either the stromal fraction generated above or from residual fibroblasts in the organoid fraction
- Thaw stromal cells or organoids at 37C quickly and resuspend in RMFC media.
- Pellet cells to remove DMSO from culture media, resuspend pellet in 10 ml RMFC.
- Plate cells and allow fibroblasts to adhere for 1-2 hours, you should see round cells that have attached to the plate and become less refractile.
- Remove non-adherent cells and discard (or use to generate epithelial cell cultures, see below).
- Feed the adherent cells with fresh RMFC. Culture and continue feeding these cells every other day to generate cultures of primary mammary fibroblasts. You should see the cells spread out first (1-3 days) so they begin to look like fibroblasts and then after about 3-7 days it should be evident that the cells are proliferating to fill the plate…this will go faster if the plate is more rather than less confluent when the cells are initially plated.
- Cell growth will vary with patient sample but should be able to be passaged for 5-7 times before they start to senesce. Note that the cells are sensitive to confluence and grow best when they are split no more than 1:3. The cells can be quite large so expect only to get 2-3 million cells from a full but not overly confluent 15-cm plate.
Epithelial cell cultures can be generated from the organoid fraction (there are some residual organoids/epithelial cells in the stromal fraction as well).
- Starting from Step4 of the above sequence for culturing fibroblasts, collect the non-adherent cells and plate in mammary growth media.
- Cells should adhere and grow out of the organoids after 3-7 days to form colonies, do not change the media during this time; feed with additional mammary media if the media looks low after 4 days. Some cells prefer to not adhere in the culture, these are typically discarded when generating traditional HMEC (human mammary epithelial cell) cultures. Adherent cells can be grown for about 5 passages before they senesce and produce variant HMEC cells that will take over the culture. The mammary media is serum free so any residual fibroblasts in the culture will not grow out under these conditions
1X Collagenase mixture Mammary Media
1.5 mg/ml collagenase MEBM + bullet kit (Lonza)
125 U/ml hyaluronidase
1X pen/strep/fungizone
in RMFC media
DMEM-Ham’s F12 (1:1 ratio) DMEM (high glucose)
5% calf serum 10% calf serum
10 ng/ml insulin 1X pen/strep/fungizone
10 mg/ml EGF
10 mg/ml hydrocortisone
