Materials and Reagents

Materials
Flasks, plates and insert
1. T-75 flasks, vent (Corning, catalog number: 08-772-1F)
2. Corning Cell Culture 12 well plates (Corning, catalog number: 07-200-82)
3. Corning 12 mm Transwell with 3.0 μm Pore Polycarbonate Membrane Insert (Corning, catalog number: 07-200-157)
Histology
1. Bio-wraps (Leica Biosystems, catalog number: 3801090)
2. Tissue and Biopsy Cassettes (Fisher Scientific, catalog number: 15-200-403J)
Tools for Constructing a Custom Stamp
1. 5-minute epoxy (JB Weld, ClearWeld # 50112, Home Depot)
2. Round wooden disks (25 mm in diameter, 3 mm in thickness, Hobby Lobby)
3. Standard 2 mm drill bit
4. Standard 5 mm drill bit
Cell
1. Primary human mammary epithelial cells (ScienCell, catalog number: 7610)
2. MCF10A human mammary epithelial cells (Barbara Ann Karmanos Cancer Institute)
3. HMT-3522-S1 human mammary epithelial cells (Mina Bissell Laboratory, Lawrence Berkeley National Laboratory, Berkeley, CA)
4. Primary human mammary fibroblasts (ScienCell, catalog number: 7630)
Media
For primary mammary epithelial cells
1. Mammary Epithelial Cell Medium (MEpiCM, ScienCell, catalog number: 7611) supplemented with 1% Mammary Epithelial Cell Growth Supplement (MEpiCGS, catalog number: 7652) and 1% penicillin/ streptomycin (Thermo Fisher, catalog number: MT30002CI)
For MCF10A human mammary epithelial cells
1. DMEM/F12 (Thermo Fisher, catalog number: 11320033) supplemented with the MCF10A additives (Recipe 1) (Debnath et al., 2003)
For HMT-3522-S1 human mammary epithelial cells
1. DMEM/F12 (Thermo Fisher, catalog number: 11320033) supplemented with the HMT-3522-S1 additives (Recipe 2) (Vidi et al., 2013)
For primary human mammary fibroblasts
1. Fibroblast Medium (FM, ScienCell, catalog number: 2301) supplemented with 1% Fibroblast Growth Supplement (FGS, catalog number: 2352) and 1% penicillin/streptomycin
Reagents
Reagents for culturing
1. 0.05% trypsin/EDTA solution (Fisher Scientific, catalog number: 25-300-062)
2. 0.25% trypsin/EDTA solution (Fisher Scientific, catalog number: 15-050-057)
3. Soybean trypsin inhibitor (10 mg/ml, Thermo Fisher, catalog number: 17075-029)
4. Dispase I (2mg [20U] Sigma-Aldrich, catalog number: D4818)
5. 0.5 M EDTA, pH 8.0 (Thermo Fisher, catalog number: AM9260G)
6. PBS (Thermo Fisher, catalog number: SH3037803)
7. EGF (Sigma-Aldrich, catalog number: E-9644)
8. Hydrocortisone (Sigma-Aldrich, catalog number: H-0888)
9. Cholera toxin (Sigma-Aldrich, catalog number: C-8052)
10. Horse serum (Thermo Fisher, catalog number: 16050122)
11. Prolactin (Sigma-Aldrich, catalog number: L6520)
12. β-estradiol (Sigma-Aldrich, catalog number: C-8052)
13. Sodium selenite (Sigma-Aldrich, catalog number: 214485-5G)
14. Transferrin (Sigma-Aldrich, catalog number: T2252)
15. HMT-3522-S1 additives (see Recipes)
16. MCF10A additives (see Recipes)
17. Phosphate buffered saline (PBS) (see Recipes)
18. Acellular layer matrix (see Recipes)
19. Cellular layer matrix (see Recipes)
20. Paraformaldehyde (see Recipes)
Reagents for ECM
1. Matrigel (~10 mg/ml, growth factor reduced, Corning, catalog number: 354230)
2. Corning Collagen I, Rat Tail (3-4 mg/ml, Corning, catalog number: CB-40236)
3. DMEM/F12 (10x), made from powder (Thermo Fisher, catalog number: 12500062)
4. Sodium Bicarbonate 7.5% solution (50x, Thermo Fisher, catalog number: 25080094)
5. FBS (JR Scientific, catalog number: 43603-500 [Research grade])
6. L-Glutamine (200 mM [50x], Thermo Fisher catalog number: 25030081)
Reagents for Histology
1. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148-500G)

Equipment

Hand drill (De Walt, model: #dcd7916 20v 1/2" chuck, Home Depot)
Stainless steel rods (Uxcell, 30 mm x 2.5 mm, Amazon.com)
4" cast iron drill press vice (Irwin, model: #226340, Home Depot)
37 °C water bath
37 °C humidified incubator with 5% CO₂
Cell culture benchtop centrifuge
Hemocytometer
Micropipettes (P20, P200 and P1000)
Pipette aid
Microscope with a color CCD camera

Procedure

Cell culture and maintenance
Primary Human Mammary Epithelial cells
Primary human mammary epithelial cells (ScienCell) were isolated from human breast and cryopreserved at passage one.
1. Prepare a poly-L-lysine (2 μg/cm²⁾-coated T-75 flask. To obtain a 2 μg/cm² poly-L-lysine-coated culture vessel, add 10 ml of sterile water to a T-75 flask and then add 15 μl of poly-L-lysine stock solution (10 mg/ml). Leave the flask in a 37 °C incubator overnight. Rinse the flask.
2. Thaw a frozen vial of cells (> 5 x 10⁵ cells) in a 37 °C water bath, and dispense the contents of the vial into the poly-L-lysine-coated flask.
  Note: Dilution and centrifugation of cells after thawing are not recommended.
3. Gently rock the flask to distribute cells evenly. Return the culture vessel to the incubator, and leave the culture undisturbed for at least 16 h after initiation.
4. Replace medium the next day to remove residual DMSO and unattached cells.
5. Thereafter, change the medium every three days, until the culture is approximately 70% confluent. Once the culture reaches 70% confluence, change the medium every other day until the culture is approximately 90% confluent.
6. Subculture when cells reach 90% confluence. Gently rinse the flask with PBS and add 4 ml 0.25% Trypsin/EDTA solution into the flask. Gently rock the flask to ensure complete coverage of cells by Trypsin/EDTA solution, return the flask to the 37 °C incubator and incubate it for 2-3 min or until cells have detached.
7. After cells have completely detached from the flask, add 180 μl of soybean trypsin inhibitor to the flask and transfer detached cells to a 15 ml centrifuge tube.
8. Centrifuge the tube at 400 x g for 5 min and discard the supernatant. Resuspend the cell pellet in culture medium and plate them in new poly-L-lysine-coated flasks at the seeding density of 5,000 cells/cm².
9. Cells can undergo ten population doublings (early passages are recommended).
MCF10A Human Mammary Epithelial cells
MCF10A cells were collected from human fibrocystic breast tissue and went through spontaneous immortalization. MCF10A cells maintain many characteristics of normal cells, including (a) the inability to form tumors in nude mice; (b) dependence on growth factors and hormones for growth and survival; and (c) lack of anchorage-independent growth (Soule et al., 1990). Culturing of MCF10A cells follows the protocol described by Debnath et al. (2003) with slight modifications.
1. Thaw a frozen vial of cells (1 x 10⁶ cells) in a 37 °C water bath and dispense the contents of the vial into a T-75 flask containing 10 ml culture medium.
2. Gently rock the flask to distribute cells evenly. Return the culture vessel to the incubator and leave the culture undisturbed for at least 16 h after initiation.
3. Replace medium the next day to remove residual DMSO and unattached cells.
4. Change the medium every three days until the culture is approximately 70% confluent. Once cells reach 70% confluence, change the medium every other day until the culture is approximately 90% confluent.
5. Subculture when cells reach 90% confluence. Gently rinse the flask with PBS and add 4 ml 0.25% Trypsin/EDTA solution into flask. Gently rock the flask to ensure complete coverage of cells by Trypsin/EDTA solution, return the flask to the 37 °C incubator and incubate it for 2-3 min or cells have completely detached.
6. Add 180 μl of soybean trypsin inhibitor to the flask and transfer detached cells to a 15 ml centrifuge tube.
7. Centrifuge the tube at 400 x g for 5 min and discard the supernatant. Resuspend cell pellets in culture medium and plate them in new flasks at a 1:3 ratio.
8. Cells can undergo 35 passages (early passages are recommended).
HMT-3522 S1 Human Mammary Epithelial cells
HMT-3522 S1 was derived from a benign mammary fibrocystic lesion and became spontaneously immortalized in culture (Briand et al., 1987; Moyret et al., 1994; Rizki et al., 2008; Vidi et al., 2013). S1 cells retain non-malignant characteristics, requiring EGF to grow in culture and being unable to form tumors in nude mice (Briand et al., 1987; Rizki et al., 2008; Vidi et al., 2013).
Note: The use of S1 cells is restricted to passages below 60 because of genotypic drift at higher passages (Rizki et al., 2008; Vidi et al., 2013). Culturing of HMT-3522 S1 cells follows the protocol described by Vidi et al. (2013) with slight modifications.
1. Thaw a frozen vial of cells (1 x 10⁶ cells) in a 37 °C water bath, and dispense the contents of the vial into a centrifuge tube with 5 ml of growth medium.
2. Centrifuge the tube at 400 x g for 5 min.
3. Remove the supernatant and resuspend the cell pellet in 3 ml of fresh growth medium.
4. Add 7 ml fresh growth medium into a T-75 flask and transfer the whole cell suspension.
5. Return the culture vessel to the incubator and leave the culture undisturbed for two days after initiation.
6. Change the medium every other day until colonies start forming rounded islands (the edges of the colonies become smooth).
  Note: This phenomenon usually takes place when the culture reaches ~60% confluence (6~10 days after plating).
7. Subculture when cells reach 60% confluence. Rinse the cells with 1 ml 0.25% Trypsin/EDTA solution and add 1 ml 0.25% Trypsin/EDTA solution into the flask. Gently rock the flask to ensure complete coverage of cells by Trypsin/EDTA solution, return the flask to the incubator and incubate it at 37 °C for 2-3 min.
8. Add 180 μl of soybean trypsin inhibitor to the flask and transfer detached cells to a 15 ml centrifuge tube.
9. Centrifuge the tube at 400 x g for 5 min and then discard the supernatant.
10. Resuspend cells in 3 ml culture medium and count cell density.
11. Place 10 ml growth medium into a T-75 flask.
12. Seed 1.5 x 10⁶ cells in a T-75 flask (2 x 10⁴ cells/cm²).
13. Cells can be used until 60 passages (early passages are recommended).
Primary Human Mammary Fibroblasts
Primary human mammary fibroblasts (ScienCell) were isolated from human breast and cryopreserved at passage one.
1. Prepare a poly-L-lysine (2 μg/cm²)-coated T-75 flask as mentioned above.
2. Thaw a frozen vial of cells (> 5 x 10⁵ cells) in a 37 °C water bath, and dispense the contents of the vial into the poly-L-lysine-coated flask. Gently rock the flask to distribute cells evenly. Return the culture vessel to the incubator and leave the culture undisturbed for at least 16 h after initiation.
3. Replace medium the next day to remove residual DMSO and unattached cells.
4. Change the medium every three days thereafter, until the culture is approximately 70% confluent. Once cells reach 70% confluence, change the medium every other day until the culture is approximately 90% confluent.
5. Subculture when cells reach 90% confluence. Rinse the cells with PBS and add 10 ml of 0.05% Trypsin/EDTA solution into flask. Gently rock the flask to ensure complete coverage of cells by Trypsin/EDTA solution, return the flask to the incubator and incubate it at 37 °C for 2-3 min. Add 180 μl of soybean trypsin inhibitor to the flask and transfer detached cells to a 15 ml centrifuge tube.
6. Centrifuge the tube at 400 x g for 5 min and discard the supernatant. Resuspend the cells in culture medium and plate them in new poly-L-lysine-coated flasks at the seeding density of 5,000 cells/cm².
7. Cells can undergo 15 population doublings.
Construction of a micropatterning stamp (Figure 1)

Figure 1. Construction of micropatterning stamp

A custom stamp for micropatterning the ECM in a 12-well plate insert is constructed as follows:
1. To find the center of a round wooden disk, draw two parallel chords with equal length across the circle with a pencil. Draw an “X” that links the ends of the chords diagonally. Mark the intersection of the “X” as the center of the circle.
2. Using a pencil, mark four 2 mm-spots on the disk at 5 mm from the center, 2 mm apart from each other.
3. Secure the disk horizontally in a drill press vice.
4. Using a hand drill in conjunction with 2 mm-drill bit, drill four holes through the spots marked on the disk.
5. Using a hand drill in conjunction with 5 mm-drill bit, drill two access holes on the opposite ends of the disk.
6. Remove the disk from the drill press vice.
7. Insert 2.5-mm stainless steel rods into the 2 mm-holes on the disk. This provides a press fit for rods to hold them in place until they are secured with epoxy.
8. Adjust the rods so that the 9 mm-ends of the rods remain above the top surface of the disk and 13 mm-ends of the rods protrude from the bottom surface of the disk.
9. Mix and apply a small amount of epoxy to the perimeter of the rods on the top side of the disk where they meet the disk to secure them in place. Air-dry the stamp as recommended on the package of the epoxy.
10. Clean the metal rods of the stamp lightly with 70% alcohol, and UV-irradiate the stamp for 10-20 min in a tissue culture hood for sterilization before use.
Preparation of 3D organotypic co-culture (Figure 2)

Figure 2. Flow Chart of 3D Organotypic Co-culture of mammary epithelial cells and fibroblasts

Day 1

Preparation of acellular layer
1. Prepare a 12-well plate.
2. Place a transwell insert into each well.
3. Adjust the concentration of type I collagen to 2 mg/ml in PBS and the pH to 7.4 with sterile 1 M NaOH.
4. To make the acellular layer, add 10x DMEM/F12, FBS, L-Glutamine, N-bicarbonate and type I collagen (2 mg/ml) in this order into a centrifuge tube on ice (Recipe 4).
5. Mix gently and readjust the pH to 7.4 with sterile 1 M NaOH.
6. Place 200 μl of gel into each insert.
7. Leave the plate undisturbed at room temperature in a tissue culture hood for 30 min during polymerization of collagen.
Preparation of fibroblast suspension
1. Grow primary mammary fibroblasts in three T-175 flasks.
2. Trypsinize and combine all dissociated cells in a 50 ml centrifuge tube.
3. Centrifuge, wash and resuspend the pellet in 1 ml of fibroblast growth medium.
4. Count the cell density and adjust it to 1 x 10⁸/ml with fibroblast medium.
Preparation of cellular layer
1. Adjust the concentration of type I collagen to 2 mg/ml in PBS and the pH to 7.4 with sterile 1 M NaOH.
2. Add 10x DMEM/F12, FBS, L-Glutamine, N-bicarbonate and type I collagen (2 mg/ml) in this order into a centrifuge tube on ice (Recipe 4).
3. Mix gently and adjust the pH to 7.4 with sterile 1 M NaOH.
4. Add Matrigel and fibroblasts.
5. Place 1.0 ml gel into each insert (1x10⁷ fibroblasts/sample).
6. Place a custom stamp into the gel (Figure 3, top left).
  
  Figure 3. Scheme of organotypic 3D ECM co-culture of primary mammary fibroblasts (MFs) and mammary epithelial cells (MECs). (Left) Schematic representation of experimental design of organotypic 3D ECM co-culture, where MECs and MFs are grown in distinct locales of the discontinuous ECM with the help of a micropatterning stamp on top of Collagen I matrix. (Right top) Image of the custom-made stamps. (Right bottom) Representative image of Hematoxylin and Eosin stained paraffin-embedded section of a co-culture showing the histomorphology of MFs and MECs at 40x and 200x magnifications. Scale bar: 50 µm.
7. Leave the plate undisturbed for 30-45 min in the tissue culture incubator (37 °C, 5% CO₂) during polymerization of the matrix.
8. Add the fibroblast growth medium through the access holes of the stamp, ~4 ml into the bottom of the wells and ~1 ml into the insert.
9. Leave the plate in the tissue culture incubator (37 °C, 5% CO₂) for one day until the matrix is manually dislodged from the inner wall of the insert.
Preparation of 3D mammary acini
Matrigel Coating and Matrigel Suspension
1. Thaw Matrigel at 4 °C overnight and keep it on ice.
2. Coat a 12-well culture plate with ~500 μl Matrigel per well. Incubate for 15-30 min in a 37 °C tissue culture incubator to allow polymerization of Matrigel.
3. In a 15 ml centrifuge tube, prepare 4% Matrigel suspension in the mammary epithelial cell growth medium.
Preparation of Mammary Acini
Note: Normal or non-malignant mammary epithelial cells form mammary acinar-like structures)
1. Trypsinize, wash and resuspend mammary epithelial cells (primary mammary epithelial cells, MCF10A cells or HMT-3522 S1 cells) in 1 ml mammary epithelial cell growth medium.
2. Count the cell density and transfer the volume of the cell suspension to have 1 x 10⁵ cells/sample in each well of a 12 well plate (0.26 x 10⁵ cells/cm²).
3. Centrifuge to collect the cell pellet, and resuspend cells in 1 ml 4% Matrigel suspension per sample.
4. Place the cell suspension onto Matrigel coat.
5. Leave the plate in the tissue culture incubator (37 °C, 5% CO₂) for six days until spheroid structures are formed.

Day 2

Dislodging collagen 1 (Acellular and Cellular) matrix (Figure 4)
1. Carefully lift up and remove the stamp from the co-culture.
2. Remove the growth medium.
3. Using a sterile glass Pasteur pipette, outline the circumference of the matrix for 2-3 times to dislodge the matrix from the inner wall of the insert.
  Note: Do not pierce the transwell membrane.
4. Place the stamp back to fit into the grooves.
5. Add 1 ml fibroblast growth medium into the insert.
6. Leave the plate in the tissue culture incubator (37 °C, 5% CO₂) for five days. The matrix will become contracted over the next few days. The medium in the bottom of the well is replaced daily (no change of the medium in the insert).
  
  Figure 4. Dislodging collagen I (COL1, acellular and cellular) matrix
Day 7
1. Combining the basement membrane harboring mammary acini
  Dissociation of Mammary Acini from Matrigel
  1. Dissolve Dispase I (2 mg, 20 U) in 200 μl PBS to make the stock solution (10 mg/ml, 100 U/ml). Further dilute Dispase I solution to make the working solution (0.2 mg/ml, 2 U/ml).
  2. Remove the Matrigel suspension from the mammary epithelial culture in the 3D matrix. Wash with PBS.
  3. Add 1 ml working solution of Dispase I (2 U/ml). Incubate for 30 min in a 37 °C tissue culture incubator for enzymatic digestion of Matrigel.
  4. Add 20 μl 0.5 M EDTA (pH 8.0, final concentration of 1 mM) to inactivate Dispase I.
  5. Collect the digest in a 15 ml centrifuge tube. Centrifuge and wash the cell pellet in PBS 2~3 times.
  6. Resuspend the pellet (~2 x 10⁴ acini) in 500 μl Matrigel and keep it on ice
  Addition of Mammary Acini in Matrigel to Collagen Matrix (Figure 5)
  1. Carefully remove the stamp.
  2. Remove the growth medium from the insert.
  3. Into four grooves formed after removing the stamp, transfer ~100 μl/groove Matrigel/mammary acini mixture. Place the remaining mixture (~100 μl) on the surface of the cellular layer. Incubate for 30 min in a 37 °C tissue culture incubator for polymerization of Matrigel (Figure 5).
  4. Prepare fibroblast growth medium/mammary epithelial cell growth medium (50:50) mixture and add ~4 ml into the bottom of the wells and ~1 ml into the insert.
  5. For the analysis of drug treatments, different drugs can be added to the growth medium.
  6. Leave the plate in the tissue culture incubator (37 °C, 5% CO₂) for seven days. Change the medium daily.
    
    Figure 5. Adding mammary acini/Matrigel mixture to the collagen matrix
Day 14
1. Harvesting the co-culture for paraffin-embedding
  1. Fix the co-cultures with 4% paraformaldehyde at 4 °C for 2~3 h. Wash the cultures in PBS at RT for 10 min.
    Note: Do not fix for too long. Over fixation will destroy certain antigens.
  2. Flip the insert and cut out the membrane. Gently push out the co-culture from the insert into the well plate. Add PBS to cover the culture.
  3. Place a Bio-Wrap inside a tissue cassette. Position the co-culture face-down on the Bio-Wrap. Fold the Bio-Wrap to wrap the co-culture and close the cassette.
  4. Put the cassettes in 70% EtOH and keep at 4 °C until processing for paraffin-embedding.
  5. Section paraffin-blocks at 10 μm and analyze the sections by immunohistochemistry (Figure 3, bottom right).

Recipes

MCF10A additives
Reagent
Final Concentration
Horse serum
5%
Penicillin/streptomycin
1%
Insulin
10 μg/ml
EGF
20 ng/ml
Hydrocortisone
0.5 μg/ml
Cholera toxin
100 ng/ml
HMT-3522-S1 additives
Reagent
Final Concentration
Prolactin
5 μg/ml
Insulin
250 ng/ml
EGF
5 ng/ml
Hydrocortisone
1.4 μM
β-estradiol
0.1 nM
Sodium selenite
2.6 ng/ml
Transferrin
10 μg/ml
Phosphate buffered saline (PBS)
Reagent
Final Concentration
NaCl
137 mM
KCl
2.7 mM
Na₂HPO₄
10 mM
KH₂PO₄
1.8 mM
Adjust pH to 7.4 and autoclave to sterilize (121 °C, 15 min)
Store PBS at 4 °C
Acellular layer matrix (Final concentration of 1 mg/ml collagen, total 1.8 ml for 6 samples)
Reagent
Volume
10x DMEM/F12
180 μl
FBS
18 μl
L-Glutamine
18 μl
Na-bicarbonate
18 μl
Type I collagen (2 mg/ml, pH 7.4)
900 μl
Sterile water
Up to 1.8 ml
Add in this order. Keep the mixture on ice until use. Mix gently and readjust the pH to 7.4 with sterile 1 M NaOH
Cellular layer matrix (Final concentration of ~1 mg/ml collagen + ~3 mg/ml Matrigel, total 10 ml for 6 samples)
Reagent
Volume
10x DMEM/F12
1.0 ml
FBS
100 μl
L-Glutamine
100 μl
Na-bicarbonate
100 μl
Type I collagen (2 mg/ml, pH 7.4)
5.0 ml
Readjust the pH to 7.4 with sterile 1 M NaOH.
Matrigel
2.5 ml
Fibroblasts (1 x 10⁸/ml)
1.0 ml
Sterile Water
Up to 10 ml
Add in this order. Keep the mixture on ice until use
Paraformaldehyde (4%, 1 L)
1. Add 800 ml of 1x PBS to a glass beaker on a stir plate and heat to ~60 °C while stirring
2. Add 40 g of paraformaldehyde powder and stir
3. Add 1 N NaOH dropwise until the solution becomes clear
4. Once the paraformaldehyde is dissolved, cool down the solution to the room temperature
5. Recheck the pH, and adjust it with 1 N HCl or NaOH to ~pH 6.9
6. Adjust the volume of the solution to 1 L with 1x PBS
7. Store the solution at 2-8 °C for up to one month

Acknowledgments

We thank Robert Del Chiaro for constructing custom micropatterning stamps; Allen Schroering in the Histology Core, UT, and all the staffs in the Department of Pathology, UT, for preparation and evaluation of histological specimens. This work was supported by the startup fund from University of Toledo Health Science Campus, College of Medicine and Life Sciences, Department of Cancer Biology to S.F; Ohio Cancer Research Grant (Project #: 5017) to S.F; Medical Research Society (Toledo Foundation) Award to S.F; and American Cancer Society Research Scholar Grant (RSG-18-238-01-CSM) to S.F.
This protocol was adapted from Debnath et al. (2003) for the method to culture MCF10A cells; Vidi et al. (2013) for the method to culture HMT-3522 S1 cells; and modified from Kalabis et al. (2012) for the method of organotypic 3D culture.

Competing interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Ethics

All animal experiments conformed to The Guide for the Care and Use of Laboratory Animals (National Research Council, National Academy Press, Washington, D.C., 2010) and were performed with the approval of the Institutional Animal Care and Use Committee of the University of Toledo, Toledo, OH (Protocol No: 108658).

References

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