The Cleared Mammary Fat Pad Transplantation Assay for Mammary Epithelial Organogenesis

1. Euthanize donor mice per institution guidelines. Sterilize each mouse with 70% ethanol.

2. Remove the mammary glands and place them into a tissue-culture plate containing DMEM/F12.

• In mice, there are five pairs of mammary glands (Fig. 1). The thoracic (#3), abdominal (#4), and inguinal (#5) pairs can be harvested for epithelial cell dissociation.

3. Remove the lymph nodes from each of the abdominal (#4) mammary glands.

4. In a laminar cell-culture hood, chop the tissues for 5–10 min with two scalpels on a tissue-culture plate.

5. Transfer the minced tissues to 10-mL conical tube containing ∼15 mL of collagenase solution. Place the tube on a shaker at 37°C for 30 min.

• This volume of collagenase solution is sufficient for up to 10 glands.

6. Centrifuge the tube at 550g for 10 min at 25°C.

• After centrifugation, the tube will have three layers: a fatty layer on top, an aqueous layer in the middle, and a pellet of cells and tissue fragments on the bottom.

7. Aspirate the fatty and aqueous layers, and resuspend the cell pellet in 10 mL of DMEM/F12. Centrifuge the tube at 550g for 5 min at 25°C. Repeat this step to remove all remaining traces of fat and enzyme.

8. Perform six rounds of differential centrifugation as follows.

• i. Aspirate the supernatant.

• ii. Resuspend the pellet in 10 mL of DMEM/F12.

• iii. Pulse the cells in the centrifuge by spinning until the centrifuge reaches 550g, then stopping the spin.

• Differential centrifugation will deplete dissociated cells such as stroma and immune cells present in the mammary gland and enrich for small epithelial tissue fragments called “organoids.” For studies where these supporting cells are of interest, omit differential centrifugations.

9. Wash the organoids in 5 mL of HBSS and centrifuge at 550g for 5 min.

10. Resuspend the organoids in 2 mL of 0.05% trypsin/EDTA and incubate at 37°C for 10 min to dissociate cells.

11. (Optional) Near completion of the 10-min digestion (Step 10), transfer the digested organoids onto a low-adhesion plate and examine them under a microscope to confirm dissociation of the cells from the organoids.

12. Terminate the trypsinization using 2 mL of 2% FBS/HBSS.

13. Pellet the cells at 550g for 5 min and resuspend in 2 mL of HBSS.

14. Add 40 µL of 1 mg/mL DNase I and incubate the cells at room temperature for 3–5 min.

15. Pellet the cells again and resuspend in 1 mL of primary growth medium. Determine the number of cells by counting using a hemocytometer.

16. Resuspend the desired cell number (ranging from 10³ to 10⁶) in 10 µL of Matrigel-GFR: Primary Growth Medium 1:1 (v/v). Keep the Matrigel:medium mixture on ice until transplantation.

• Each cleared MFP will be injected with a 10-µL volume containing cells.

17. Obtain appropriate recipient mice aged 21–28 d. Anesthetize the mice with an intraperitoneal injection of 200–400 µL of Avertin (2.5%, diluted in saline).

18. Place a mouse on a plastic board with legs taped down. Shave the fur on the abdomen (unless the mouse is hairless) and wipe the abdominal skin with betadine and alcohol.

19. Make a 1-cm incision with scissors at the midline longitudinally starting at the belly button.

20. Make two 0.5-cm incisions from the bottom of the midline incision to the hip region just above the leg. Pull the skin back from the peritoneum using forceps to expose the mammary gland and clamp down with an inverted tweezer.

21. To prevent excessive bleeding, use a handheld cauterizing device to sever three vessels before clearing the MFPs: Cauterize the mammary artery running between the #4 and #5 MFPs, as well as the two blood vessels that form a triangle around the proximal lymph node.

22. Cut the bridge between the #4 and #5 mammary glands with the cauterizing device to severe the connection between them.

• This will prevent the epithelium from the #5 gland from growing into the #4 experimental MFP.

23. To remove the region of the MFP between the nipple and just after the proximal lymph node, first make one cut across the MFP just after the lymph node. Next, remove the portion of MFP between the lymph node and the mammary artery (Fig. 2).

• (Optional) The tissue piece removed from the mammary glands can be spread on a microscope slide and stained with Carmine Alum to confirm that the entire rudimentary epithelial bud has been removed.

24. Load the cell/Matrigel mixture from Step 16 into a syringe for injection. Inject the cells into the remaining portion of the epithelium-free MFP between the inguinal lymph node and the midline.

25. Close the skin incisions with wound autoclips and surgical glue.

26. Repeat the procedure, if desired, on the contralateral mammary gland.

27. Place the mouse on a warming pad and monitor every 10–15 min until it regains consciousness. Monitor the mouse according to institutional guidelines for up to 1 wk for signs of infection or other surgical complications.