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Primary Mammary Epithelial Isolation\

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.

 

PRECOAT ALL PIPETTES and FALCON TUBES with 5% BSA-PBS

 

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).