Primary Mammary Epithelial Isolation
Mince glands with two razor blades on sterile surface.
Make collagenase solution and prewarm at 37C (shake occasionally)- filter sterilize after the collagenase has gone into solution through a .2-micron filter.� Put solution into fifty ml falcon. Must make this media fresh (collagenase will inactivate at high temperatures and over long periods of time):
����������� Collagenase Solution:��� 0.2% trypsin
����������������������������������������������� 0.2 % Collagenase A
����������������������������������������������� 5% FCS (fetal calf serum)
����������������������������������������������� 5ug/ml Gentamycin
����������������������������������������������� 47.5 mls DMEM/F12 media
Shake minced tissue in filtered collagenase solution at 100 rpm/37C for 30 minutes.
Spin@ 1500 rpm for 10mins.�
1. The top layer is going to be the fatty layer, but it will often contain uncollected organoids, so collect 10mls of the top layer in a 15ml falcon.� Pipette it up and down to break it up and free organoids.
2. The pellet at the bottom will be your epithelial cells, fibroblasts, etc.� Remove all the rest of the media.� Resuspend the pellet at the bottom in 10 mls of 0% DMEM/F12 (0% means no FCS in the media � FCS makes the media too dense to get separation) and put in 15 ml falcon tube.
Take the two 15 ml falcon tubes and spin them @1500rpm for 10 minutes.
Remove the media from the two falcon tubes and combine the two pellets by resuspending one then the other in the same 4 mls of 0% DMEM/F12.� Now you have 1 15ml falcon of organoids.� Add 40ul of DNase (2U/ml) � shake by hand for 2-5 minutes at room temperature (until clumps are broken up) � add 6 mls of 0% DMEM/F12.
Spin@ 1500 rpm for 10 mins (to remove DNase).
Discard supernatant � resuspend pellet in 10 mls of 0% DMEM/F12.
Spin@ 1500 for a brief second/pulse�cut the machine and hit the brakes when it gets to 1500. �You will have a pellet of epithelial organoids.
If you want a fibroblast culture � they are in the supernatant at this step and you can put them into a dish and culture them.� Otherwise, aspirate them off with the media.
You MUST clean up the epithelial organoids by repeating the pulse 4-10 times.� Each time monitor the composition of the pellet by examining 10ul of the resuspended pellet under the microscope.� Look for mainly organoids and a few single cells.� Single cells are mainly the fibroblasts so you want to get rid of them.� Some epithelial cells are in the single cell form, so you do not have to completely remove all single cells.� 10 pulses are generally more than sufficient to get a clean epithelial culture.
Resuspend the pellet at the desired concentration into Growth Medium:
����������� 100mls of Growth Medium: ���� 100ul of ITS (1000X dilution)
����������������������������������������������������������� 5ul of EGF (100ug/ml)
����������������������������������������������������������� 5 mls of FCS
����������������������������������������������������������� 100ul of Gentamycin (50mg/ml)
����������������������������������������������������������� 1 ml of Pen/Strep (100X dilution)
����������������������������������������������������������� 94 mls of DMEM/F12 media
����������������������������������������������������������� FUNGIZONE (100X dilution) OPTIONAL (primary cells do not seem to mind Fungizone, I suggest it because primary preps are pretty messy and are easily contaminated, especially if you dissect in the open air.� If you do not want to use Fungizone in your media, and this is understandable, I would dissect the mice in a hood and dip them in ethanol including the tail before dissection, then also make sure to sterilize all tools before hand, avoid mouse hair and/or any other contamination in the prep).
Change media next day and every two days after.� Passage the cells at 1:1 or 1:2 split ratio for the first two passages.� Then you can get more liberal and move up to 1:5 split.� They should survive 5-7 passages before reaching senescence.�
Use dispase to passage or a 2mg/ml collagenase in DMEM/F12 media solution.� Leave 20-30 mins at 37C.� Tap culture to release cells from plate.� Can also pipette up and down.� Do not want to go over 30 mins, as you will start to seriously damage the cells.
If you are looking to create a monolayer of cells on plastic you need to make sure to plate the cells at a high density the first night.� It is better to go too dense because you can remove the supernatant the next day and save any unattached cells by plating them on their own plate at high density.� Low-density cultures do not do well or maintain proper culture morphology.
You may want to coat the plate before plating with a thin layer of Collagen I.� We use Vitrogen (product name) and dilute 1ml Vitrogen in 44mls of PBS.� We add 3 mls of this mixture for every 25-cm2 surface area of culture dish.� It must gel over night in an airtight case at 4C.� Before use, aspirate off the excess PBS and the bottom of the culture dish will have a thin layer of Vitrogen (collagen I).