Decellularizing Mammary Fat Pads: Deriving Extracellular Matrix Hydrogels from Mammary Fat Pads

Overview

This video describes a technique of deriving extracellular matrix hydrogels by decellularizing murine mammary fat pads following ex vivo irradiation for in vitro studies. This hydrogel helps to mimic the in vivo breast tissue environment post-radiation treatment to study tumor cell behavior.

Protocol

1. Decellularization

NOTE: This procedure was adapted from previously published methods focused on adipose decellularization, which included the sodium deoxycholate ionic detergent rather than sodium dodecyl sulfate to remove DNA efficiently.

1. On day 1, remove frozen Mammary Fat pads or MFPs from -80 °C and thaw at room temperature.
2. Once thawed, dry MFPs briefly on a delicate task wipe. Weigh the MFPs using an analytical scale.
3. Using a pair of forceps with scissors or a scalpel, divide tissue up into 3 mm x 3 mm x 3 mm samples for study of the intact ECM and the remaining tissue for hydrogel production.
   NOTE: The number of samples is dependent on the number of testing methods, e.g. the collection of two samples is described below: one for paraffin embedding (step 1.5) and one for freezing in cryostat embedding medium, if desired (see the Table of Materials and step 1.6).
4. Weigh the tissues. If embedding in paraffin for sectioning, continue to step 1.5. If freezing in cryostat embedding medium for sectioning, continue to step 1.6.
5. In a chemical hood, submerge the tissue in 10% neutral buffered formalin (NBF) (see the Table of Materials) for 24 h at 4 °C. Wash 3 times in PBS for 5 min each. Submerge the tissue in 30% sucrose for 48 h at 4 °C.
   1. Weigh the tissue now that this piece has been removed. Continue to step 1.6.
6. In a chemical hood, place MFP pieces in a labeled cassette prepped with cryostat embedding medium. Add more cryostat embedding medium to cover the tissue.
   1. Place the cassette into a beaker of 2-methylbutane (see the Table of Materials) that is pre-cooled with liquid nitrogen. The beaker should have enough 2-methylbutane to cover the bottom but not enough to submerge the cassette because the cryostat embedding medium should not touch the 2-methylbutane. Let the cassette sit in the 2-methylbutane until the cryostat embedding medium freezes and becomes opaque.
   2. Wrap the cassette(s) in foil, label, and leave at -80 °C until used for sectioning.
      NOTE: Tissues placed immediately in cryostat embedding medium were sectioned at 5 μm while tissues incubated in sucrose were sectioned at 30 μm to retain adipocyte morphology.
7. Use forceps to manually massage the remaining tissue.
   NOTE: Tissue pieces may also be placed in 10% NBF for 24–48 h, rinsed in PBS, and left in 70% ethanol until embedding in paraffin. Following embedding, 5 μm sections can be used for hematoxylin and eosin (H & E) staining (see section 2 below).
8. Place the MFPs in 6 cm dishes with 5 mL 0.02% trypsin/0.05% EDTA solution. Incubate at 37 °C for 1 h. Spray and wipe the dishes with 70% ethanol before placing in the incubator.
9. Use 0.7 mm strainers to wash the MFPs with deionized (DI) water by pouring water over the tissue three times. Use forceps to manually massage the tissue in between washes.
10. Briefly dry tissue on a delicate task wipe and weigh. Place tissues in a pre-autoclaved beaker containing an appropriately sized stir bar. Cover tissues with 60 mL of 3% t-octylphenoxypolyethoxyethanol (see the Table of Materials) per 1 g of tissue and stir for 1 h at room temperature. Use a minimum of 20 mL.
11. Dump tissue and contents into a strainer. Rinse the beaker with DI water and pour onto tissues. Repeat two more times. Use forceps to manually massage the tissue in between rinses.
12. Briefly dry tissue on a delicate task wipe and weigh. Place tissues and stir bars back in the same beakers, and cover with 60 mL of 4% deoxycholic acid per 1 g of tissue. Stir for 1 h at room temperature. Use a minimum of 20 mL.
13. Dump tissue and contents into a mesh strainer. Rinse the beaker with DI water and pour onto tissues. Repeat two more times. Use forceps to manually massage the tissue in between rinses.
14. Briefly dry tissue on a delicate task wipe and weigh.
15. Place tissues in the same beaker with fresh DI water supplemented with 1% penicillin-streptomycin. Cover tightly with paraffin film. Leave overnight at 4 °C.
16. Wash strainers and beakers for use the following day.
17. On day 2, drain beaker contents into a strainer. Briefly dry tissue on a delicate task wipe and weigh.
18. Place MFPs in the same beaker with an appropriately sized stir bar. Cover with 60 mL 4% ethanol/0.1% peracetic acid solution per 1 g of tissue. Use a minimum of 20 mL. Stir for 2 h at room temperature.
19. Dump tissue and contents into a 0.7 mm strainer. Use forceps to manually massage the tissue. Place contents back into the beaker. Wash tissue by covering it with 60 mL of 1x PBS per 1 g of tissue. Use a minimum of 20 mL. Stir for 15 min at room temperature. Repeat once.
20. Dump tissue and contents into a 0.7 mm strainer. Use forceps to manually massage the tissue. Place contents back into beaker. Wash tissue by covering it with 60 mL DI water per 1 g of tissue. Use a minimum of 20 mL. Stir for 15 min at room temperature. Repeat once.
21. Briefly dry tissue on a delicate task wipe and weigh. Dump tissue and contents into a strainer. Use forceps to manually massage the tissue.
22. Place contents back into beaker. Cover tissues with 60 mL of 100% n-propanol per 1 g of tissue. Use a minimum of 20 mL. Stir for 1 h at room temperature.
23. Briefly dry tissue on a delicate task wipe and weigh. Dump tissue and contents into a 0.7 mm strainer. Use forceps to manually massage the tissue.
24. Place contents back into beaker. Wash tissue by covering it with 60 mL of DI water per 1 g of tissue. Use a minimum of 20 mL. Stir for 15 min at room temperature. Repeat three times.
25. Dump tissue and contents into a strainer. Repeat steps 2.3–2.6 to collect pieces of tissue for sucrose incubation and freezing in cryostat embedding medium.
26. Briefly dry tissue on a delicate task wipe and weigh. Place in a labeled 15 mL tube. Freeze at -80 °C overnight.

Materials

Name	Company	Catalog Number	Comments
10% Neutral Buffered Formalin, Cube with Spigot	VWR	16004-128
2-methylbutane	Alfa Aesar	19387
Bovine Serum Albumin	Sigma-Aldrich	A1933-25G
Dulbecco's phosphate-buffered saline	Gibco	14040133
Fisher Healthcare Tissue-Plus O.C.T. Compound	Fisher Scientific	23-730-571	cryostat embedding medium
Kimtech Science Kimwipes	Kimberly Clark		delicate task wipes
n-Propanol (Peroxide-Free/ Sequencing), Fisher BioReagents	Fisher Scientific	BP1130-500
PBS (10X), pH 7.4	Quality Biological, Inc.	119-069-151	Phosphate-buffered saline
Penicillin-Streptomycin	Gibco	15140-122
Peracetic acid	Sigma-Aldrich	77240-100ML
Triton x-100	Sigma-Aldrich	X100-100ML	t-Octylphenoxypolyethoxyethanol
Trypsin-EDTA (0.25%), phenol red	Gibco	25200-056
Richard-Allan Scientific Signature Series Hematoxylin 7211	Richard-Allan Scientific	7211
Whatman qualitative filter paper, Grade 4	Whatman	1004-110	grade 4 qualitative filter paper
Xylenes (Certified ACS), Fisher Chemical	Fisher Scientific	X5-5