Materials and Reagents

100-mm tissue culture Petri dish (e.g., Corning, catalog number: 353003 )
0.2-μm filters and 50 ml syringes (e.g., GVS, catalog number: FJ25ASCCA002DL01 )
No. 22 disposable scalpel blades (e.g., Swann-Morton, catalog number: 0508)
50-ml tubes (e.g., Corning, catalog number: 352070 )
15-ml tubes (e.g., Corning, catalog number: 352096 )
10-ml disposable plastic pipettes (e.g., Corning, catalog number: 357551 )
25-ml disposable plastic pipettes (e.g., Corning, catalog number: 357535 )
24-well tissue culture plates (e.g., Corning, catalog number: 353047 )
30 G insulin syringes (e.g., BD Microfine, catalog number: 324826 )
Plastic histology molds (e.g., Thermo Scientific, catalog number: 1830 )
Plastic embedding cassettes (e.g., Simport, catalog number: M492-2 )
Histology tissue molds (e.g., Simport, catalog number: M474-3 )
Microscope slides for histology (e.g., Thermo Scientific, catalog number: J1800AMNZ )
Mice: virgin females, 7–10 weeks old, inbred strain C57BL/6J (e.g., The Jackson Laboratory, catalog number: 000 664 )
Ethanol (EtOH), 70%, 95%, and 100% (e.g., VWR, catalog number: 83813 )
Phosphate-buffered saline (PBS) (e.g., Sigma-Aldrich, catalog number: D1408 )
Dulbecco’s modified Eagle medium (DMEM)/F12 (e.g., Gibco, catalog number: 21331-020 )
Bovine serum albumin (BSA) (e.g., Sigma-Aldrich, catalog number: A3608 )
Fetal bovine serum (FBS) (e.g., Sigma-Aldrich, catalog number: F0804 )
Collagenase A (e.g., Roche, catalog number: 11088793001 )
Trypsin (e.g., Dutcher Dominique, catalog number: P10-022100 )
Insulin (e.g., Sigma-Aldrich, catalog number: I6634-100MG )
Gentamicin (e.g., Sigma-Aldrich, catalog number: G1397 )
Glutamine (e.g., Gibco, catalog number: 35050-061 )
DNase I (e.g., Sigma-Aldrich, catalog number: D4527-40KU )
Dispase II (e.g., Roche, catalog number: 13 75 2000 )
Growth factor-reduced Matrigel^® (e.g., Corning, catalog number: 354230 )
Insulin-transferrin-selenium (ITS) (e.g., Gibco, catalog number: 41400-045 )
Penicillin/Streptomycin (e.g., Gibco, catalog number: 15140-122 )
FGF2 (e.g., Gibco, catalog number: PM60034 )
Prolactin (e.g., Sigma-Aldrich, catalog number: SRP4688 )
Hydrocortisone (e.g., Sigma-Aldrich, catalog number: S H6909 )
Oxytocin (e.g., Sigma-Aldrich, catalog number: O3251 )
RNeasy Micro Kit (e.g., Qiagen, catalog number: 74004 )
β-Mercaptoethanol (e.g., Sigma-Aldrich, catalog number: M6250 )
Phosphatase inhibitor cocktail II (e.g., Millipore, catalog number: 524625 )
RIPA buffer (e.g., Sigma-Aldrich, catalog number: R0278 )
Protease inhibitor cocktail I (e.g., Sigma-Aldrich, catalog number: 539131 )
Pierce Coomassie (Bradford) Protein Assay Kit (e.g., Thermo Scientific, catalog number: 23200 )
Paraformaldehyde (PFA), 32% (e.g., Electron Microscopy Sciences, catalog number: 15714 )
Low gelling temperature agarose (e.g., Sigma-Aldrich, catalog number: A9414 )
Xylene (e.g., Sigma-Aldrich, catalog number: 534056 )
Paraffin (e.g., Sigma-Aldrich, catalog number: 1071642504 )
Dissociation solution (see Recipes)
BSA solution (see Recipes)
Basal organoid medium (BOM) (see Recipes)
Morphogenesis medium (see Recipes)
Lactation medium (see Recipes)
4% PFA (see Recipes)
RNA lysis buffer (see Recipes)

Equipment

Surgical tools
Forceps (e.g., Phymep, catalog numbers: 11050-10 and 11051-10 )
Scissors (e.g., Phymep, catalog number: 14088-10 )
Dissection board (e.g., Thermo Scientific, catalog number: 36-119 )
P1000 pipette
Laminar flow hood
Fridge 4°C (e.g., Liebherr, catalog number: 7083 001-01 )
Freezer –80 °C (e.g., Thermo Scientific, catalog number: 88400V )
Liquid nitrogen tank (e.g., Air Liquide Espace 151, catalog number: 2433867 )
Shaking incubator at 37°C (e.g., Infors HT Multitron)
Centrifuge (e.g., Thermo Scientific, model: Sorvall ST40 )
Incubator for cell culture, 37°C, 5% CO₂(e.g., Thermo Scientific, model: HERAcell 150i )
Heating plate at 37°C (e.g., Techne DRI-Block DB-2A)
Microscope and camera (e.g., Olympus model: CKX41 )
NanoDrop™ (e.g., Implen Nanophotomoter NP80)
Sonicator (e.g., Diagenode Bioruptor Pico)
Incubator at 65°C (e.g., Memmert Incubator I)
Embedding workstation (e.g., Leica EG1150C )

Procedure

Isolation of mammary primary organoids
1. Dissection of a virgin mouse to harvest mammary glands (see Video 1).
  
  Video 1. Mammary gland harvesting. This video was made at Pasteur Institute. according to guidelines from the regulations of Institut Pasteur Animal Care Committees (CETEA).. on Animal Care and approved by the French legislation in compliance with European Communities Council Directives (A 75-15-01-3).
  1. Euthanize the donor mouse using an ethically approved method (e.g., cervical dislocation) and immediately proceed to mammary gland collection.
    Notes:
    1. Cervical dislocation is a common method for animal euthanasia and provides a fast and painless death. With this method, cell/tissue survival in culture is not altered if collected immediately.
    2. In the case of processing multiple mice, euthanize one animal and collect the glands immediately, then proceed to the next animal.
  2. Sanitize the ventral side of the animal by spraying 70% EtOH on the skin.
    Note: After disinfection, work inside a laminar flow hood to maintain aseptic conditions. Application of aseptic work procedures, together with the presence of antimycotic and antibiotic supplements (gentamicin in digestion solution; penicillin and streptomycin in culture medium) will prevent the occurrence of contamination.
  3. Pin the mouse by its four paws to a dissection board, with the abdomen facing upward (see Figure 1A, pins 1–4).
  4. Using forceps, tightly grasp the skin of the lower part of the abdomen at half the width (see Figure 1A, point A).
  5. Using surgical scissors, make the first incision in the skin at point A.
    Note: Be careful to incise only the skin and not rupture the underlying peritoneum.
  6. Continue to incise the skin cranially to the throat of the animal (see Figure 1A, from point A to point B).
  7. From this median line, use forceps to grasp the skin and cut toward each of the four paws (see Figure 1A, incise to join the middle line to points C, D, E or F, respectively).
  8. Using forceps and a cotton swab, gently separate the skin from the peritoneum on one side of the animal. Attach the skin to the dissection board with three pins (see Figure 1B, pins 5–7).
  9. Repeat step 8 on the other side of the animal (see Figure 1B, pins 8–10). The mammary glands are now exposed.
  10. Identify the lymph node of the mammary gland #4 (a small dense structure, round in shape; see Figure 1B, surrounded). Remove the lymph node from both glands using forceps and scissors and discard.
  11. Proceed to the harvest of the mammary glands #3 and #4. Using curved forceps, grasp the mammary glands and gently separate them from the skin and other tissues with scissors.
    Note: Carefully separate the mammary glands #3 (whitish and shiny) from the muscles (light brown ribbed structure) since this protocol does not prevent muscle contamination.
  12. Place all the collected glands in the same sterile Petri dish containing cold PBS (approximately 3 ml, previously stored at 4°C) for washing prior to tissue processing.
  13. Properly dispose of the animal corpse and continue with mechanical and enzymatic dissociation of the mammary glands.
2. Mechanical and enzymatic dissociation
1. Organoid counting
  Reminder: Work inside a laminar flow hood to maintain aseptic conditions.
  1. Draw two large crosses with a marker on a microscope slide.
  2. Take the organoid suspension and homogenize by performing five up-and-down motions with a P1000 pipette.
  3. On the reverse side of the slide, spread 10 μl solution around the center of each cross.
    Note: Use a 20-μl tip or cut the extremity of a 10-μl tip to avoid large organoids becoming trapped.
  4. Count the organoids under the microscope at 4× magnification (see Figure 1D).
    Notes:
    1. Take each quarter of the cross as a landmark to avoid double-counting of the same organoid.
    2. Organoids appear as rounded structures with a smooth perimeter. Occasionally and unavoidably, nerves and endothelium are also present. The nerves appear as rope-like structures and can be organized in bundles (see Figure 1D). The endothelium has a somewhat ragged look in comparison with the smooth-looking organoids. The minor presence of primary nerves and endothelium does not interfere with organoid lactation or involution.
    3. Count only the organoids with a diameter greater than 30–50 μm since the smaller ones may not develop properly.
  5. Calculate the average of the two counts in 10 μl solution and multiply according to the volume of BOM used to resuspend the pellet to obtain the total number of organoids.
    Note: Freshly isolated organoids can be viably frozen in a solution of FBS containing 10% DMSO for long-term storage in liquid nitrogen and later use.
1. 3D culture of mammary organoids
  1. Embedding in Matrigel^®
    Reminder: Work inside a laminar flow hood to maintain aseptic conditions. Wash the ice bucket and heating plate thoroughly with 70% EtOH prior to placement in the laminar flow hood.
    1. Thaw the Matrigel^® on ice or at 4°C.
      Notes:
      1. Matrigel^® solidifies really fast at room temperature. Always keep it on ice before use and during the plating procedure.
      2. Keep in mind that Matrigel^® thawing takes time; therefore, begin thawing prior to the procedure (2 h for a 1-ml aliquot, 6 h for a 10-ml bottle).
    2. Place a 24-well plate on ice. Calculate the number of wells needed and spread 20-μl Matrigel^® in a round patch on the bottom of each well.
      Note: Start by placing the tip containing Matrigel^® at the center of a well and expand circularly towards the edges of the well, without touching them.
    3. Incubate the 24-well plate in a cell incubator (5% CO₂) for 15 min at 37°C.
    4. In the meantime, pre-heat a heating plate to 37°C.
    5. Prepare the organoid suspension in the Matrigel^®: calculate the volume of organoid suspension required to obtain the desired number of organoids. Pipette this volume of suspension into a fresh 1.5-ml tube and centrifuge for 3 min at 400 × g.
      Note: Adjust the number of organoids per well depending on the type of experiment: 200 organoids per well for morphology and histology, 400 for gene expression, and 1000 for western blotting analysis.
    6. Carefully remove the supernatant and place the tube on ice. Subsequently, carefully resuspend the pellet in the required volume of cold Matrigel^® (50 μl per well), avoiding bubble formation. Keep on ice.
    7. Remove the 24-well plate from the cell incubator and place on the 37°C heating plate.
    8. In each Matrigel^®-precoated well, cautiously seed the suspension of organoids (in Matrigel^®) as a dome on top of the solidified Matrigel^® patch.
    9. Place the 24-well plate back in the cell incubator (5% CO₂) for 30 min at 37°C to solidify the Matrigel^® (see Figure 1D).
    10. In the meantime, pre-warm BOM at 37°C.
    11. Following incubation, carefully add 1 ml pre-heated BOM to each well and culture in the cell incubator at 37°C, 5% CO₂.
      Notes:
      1. Add medium against the edges of the well to avoid disruption of the dome.
      2. Characterization of the organoids can be performed using regular histological stains (e.g., hematoxylin & eosin) or immunostaining on day 1 post-recovery in BOM (see Figure 1E and Step B2 of the procedure).
  2. Morphogenesis with FGF2
    Reminder: Work inside a laminar flow hood to maintain aseptic conditions.
    Note: Overnight recovery is optimal for organoid culture; however, FGF2 treatment can be administered immediately after plating the organoids.
    1. Pre-heat the BOM at 37°C.
    2. Add fresh FGF2 at a final concentration of 2.5 nM to pre-heated BOM to obtain the morphogenesis medium.
    3. Aspirate the medium from the wells without touching the Matrigel^® dome and replace with 800 μl fresh morphogenesis medium.
    4. Renew all medium with fresh morphogenesis medium every 3 days, for a total of 6 days of treatment.
  3. Lactogenic differentiation with prolactin
    1. Pre-heat the BOM at 37°C.
    2. Add 1 μg/ml prolactin and 1 μg/ml hydrocortisone to the pre-heated BOM to obtain the lactation medium.
    3. Aspirate the medium from the wells without touching the Matrigel® dome and replace with 800 μl fresh lactation medium.
    4. Renew all medium with fresh lactation medium every two days, for a total of 4 days of treatment.
  4. Myoepithelial cell contraction with oxytocin
    1. Prepare fresh lactation medium, filter, and pre-heat at 37°C.
    2. Add 40 μg/ml recombinant oxytocin to the lactation medium.
    3. Aspirate the medium from the wells without touching the Matrigel^® dome and replace with 800 μl fresh lactation medium supplemented with oxytocin.
    4. Using live cell imaging, record contraction images every second for 120 s.
  5. Mimicking involution by hormonal withdrawal
    1. Pre-heat the BOM at 37°C.
    2. Aspirate the medium from the wells without touching the Matrigel^® dome and replace with 800 μl fresh BOM.
    3. Renew all medium with BOM every two days, for a total of 8 days of treatment.
  6. Replating
    Note: Use tips pre-coated with 2.5% BSA.
    1. Aspirate the supernatant and wash the wells twice with 800 μl cold PBS.
    2. Add 1 ml cold PBS and disrupt the Matrigel^® dome using an up-and-down motion with a P1000 pipette.
    3. Check for successful disintegration of the Matrigel^® under a microscope.
    4. Transfer the suspension to a 15-ml tube and add cold PBS to a total volume of 10 ml.
    5. Centrifuge for 3 min at 400 × g.
    6. Carefully remove the supernatant, resuspend the organoid pellet in fresh Matrigel^® and plate as described in B1.

Organoid processing for further analysis
Note: We suggest carefully following organoid development under the microscope before renewing the media. Morphogenesis with FGF2 should induce branching after 3–4 days of treatment, while organoids in culture with BOM only, as the negative control, should remain round. Lactogenic differentiation can be confirmed by analysis of Csn2 mRNA using qPCR, comparing organoids before and after prolactin treatment (d6 versus d10). The involution process can also be confirmed using qPCR by detecting decreased expression of Csn2 mRNA following prolactin withdrawal (d10 versus d18), or at the morphological level by the progressive disappearance of branching (see Figure 2B and Figure 3B).

Figure 2. Modeling lactation and involution-like processes in primary mammary organoids. A. Scheme depicting the experimental design. B. Morphology of primary mammary organoids during lactation and involution-like processes. Bright-field images of organoid morphology following morphogenic and lactogenic stimulation and on days 4 or 8 after hormonal withdrawal. Scale bar = 100 μm.

Figure 3. Passage of involution-like organoids. A. Scheme depicting the experimental design. PRL: Prolactin; BOM: basal organoid medium. B. Morphology of passaged organoids during the lactation and involution-like processes. Brightfield images of passage 1 (upper panel) and passage 2 (lower panel) organoids following morphogenic and lactogenic stimulation and on days 4 or 8 after hormonal withdrawal. Scale bar = 100 μm.
1. RNA isolation
  Note: Embedding in Matrigel^® does not interfere with the quality of extracted RNA.
  1. Aspirate the culture medium.
  2. Add 350 μl RLT buffer (RNeasy Micro Kit) contiaining 3.5 μl β-mercaptoethanol to each well.
  3. Disintegrate the organoid culture in lysis buffer by performing ten up-and-down motions with a P1000 pipette.
  4. Transfer the solution to a fresh 1.5-ml tube and vortex well.
    Note: Samples can be stored at –80°C until RNA extraction. To perform RNA extraction, thaw samples on ice and proceed according to the following instructions.
  5. Homogenize RNA lysates by performing ten up-and-down motions with a single-use 30 G insulin syringe.
  6. Process samples as described in the RNeasy Micro Kit booklet, starting from Step C1b.
  7. Measure the RNA concentration using a NanoDrop™.
2. Protein extraction
  Note: Embedding in Matrigel^® interferes with western blotting analysis. Follow these steps to remove the Matrigel® prior to protein extraction.
  1. Aspirate the culture medium and dissociate the 3D culture with 800 μl cold PBS supplemented with phosphatase inhibitor cocktail II.
  2. Transfer the suspension to a clean 1.5-ml tube and centrifuge for 3 min at 400 × g, 4°C.
  3. Rinse twice with PBS supplemented with phosphatase inhibitor cocktail II.
  4. Discard the supernatant and resuspend the pellet in 100 μl ice-cold ready-to-use RIPA buffer supplemented with protease inhibitor cocktail I and phosphatase inhibitor cocktail II.
    Note: Samples can be stored at –80°C until protein extraction. To perform protein extraction, thaw samples on ice and proceed according to the following instructions.
  5. Sonicate the samples twice at 4°C using a 60-kHz ultrasonic wave frequency program (30 s ON/30 s OFF).
  6. Vortex the samples, cool on ice, and repeat the sonication according to Step C2e.
  7. Centrifuge for 20 min at >10,000 × g, 4°C.
  8. Transfer the supernatant to a clean 1.5-ml tube.
  9. Measure the protein concentration using a Coomassie Protein Assay Kit.
3. Fixation and embedding for histology
  1. Aspirate the culture medium and rinse the culture twice with 800 μl cold PBS.
  2. Incubate with 800 μl 4% PFA for 30 min. Following removal of the 4% PFA, wash twice with PBS.
    Notes:
    1. Domes should be entirely covered with the solution. Add a greater volume if required.
    2. The fixed cultures can be stored in PBS at 4°C until embedding.
  3. Prepare 3% low gelling temperature agarose in PBS and melt slowly in a microwave for 1.5–2 min at 1000 W (homogenize every 30 s by hand rotation).
  4. Detach the fixed culture using the flat side of a spatula and transfer to a plastic histology mold containing melted agarose. Overlay with more agarose.
  5. After solidification of the agarose, unmold the block. Use a scalpel to remove the excess agarose surrounding the Matrigel^® dome and place in a plastic embedding cassette for histology.
  6. Proceed to sample dehydration: incubate the embedding cassettes in successive 1-h baths of 70% EtOH, 95% EtOH, 100% EtOH (twice), xylene (twice), 50% xylene-50% melted paraffin, and 100% melted paraffin.
  7. Incubate overnight at 65°C in a second bath of 100% melted paraffin.
  8. Embed in a histology tissue mold using an embedding workstation.
  9. Unmold the paraffin blocks after 24 h of solidification.
  10. Cut 5-μm sections and spread on microscope slides. Keep the slides at room temperature until further analysis.
  11. Remove the paraffin prior to any staining by successive 5-min baths of xylene (twice), 100% EtOH (twice), 95% EtOH, 70% EtOH, and H₂O.

Recipes

Dissociation solution
Note: This solution is prepared inside a laminar flow hood under aseptic conditions and does not need to be filter-sterilized.
2 mg/ml collagenase
2 mg/ml trypsin
5 μg/ml insulin
50 μg/ml gentamicin
5% FBS
2 mM glutamine
in DMEM/F12
BSA solution
Note: This solution can be filter-sterilized and reused several times when stored at 4°C.
2.5% BSA in PBS
Basal organoid medium (BOM)
Note: This solution is prepared inside a laminar flow hood under aseptic conditions and does not need to be filter-sterilized.
1× insulin-transferrin-selenium (ITS)
100 U/ml penicillin
100 μg/ml streptomycin
2 mM glutamine
in DMEM/F12
Morphogenesis medium
Note: This solution is prepared inside a laminar flow hood under aseptic conditions and does not need to be filter-sterilized.
2.5 nM FGF2 in BOM
Lactation medium
Note: This solution is prepared inside a laminar flow hood under aseptic conditions and does not need to be filter-sterilized.
1 μg/ml prolactin
1 μg/ml hydrocortisone
in BOM
4% PFA
Note: This solution is prepared inside a chemical hood and does not need to be filter-sterilized.
4% paraformaldehyde in PBS
RNA lysis buffer
Note: This solution is prepared inside a chemical hood and does not need to be filter-sterilized.
10 μl β-mercaptoethanol per 1 ml RLT lysis buffer (from the RNeasy Micro Kit; this solution can be stored for up to one month at room temperature).
Phosphatase inhibitor cocktail II
Note: This solution is prepared inside a chemical hood or on a bench and does not need to be filter-sterilized.
2 mM imidazole
1 mM sodium fluoride
1.15 mM sodium molybdate
1 mM sodium orthovanadate
4 mM sodium tartrate dihydrate
in RIPA buffer
Protease inhibitor cocktail I
Note: This solution is prepared inside a chemical hood or on a bench and does not need to be filter-sterilized.
500 μM AEBSF hydrochloride
150 nM aprotinin
1 μM protease inhibitor E-64
0.5 mM EDTA
1 μM leupeptin hemisulfate
in RIPA buffer

Acknowledgments

Work in the laboratory of HL is funded by the Pasteur, Centre National pour la Recherche the Agence Nationale de la Recherche (ANR-10-LABX-73 and ANR-16-CE13-0017- 01), Fondation ARC (PJA 20161205028 and 20181208231), Programme Barrande, and AFM-Telethon Foundation. AC was funded by postdoctoral fellowships from the Revive Consortium. EC was funded by a Ph.D. fellowship from Sorbonne Universite. ZK was funded by the Grant Agency of Masaryk University (MUNI/G/1446/2018), Mobility grant by Ministry of Education, and Youth and Sports, and by funds from the Faculty of Medicine MU to the junior researcher (ROZV/28/LF/2020). JS was funded by the P-Pool (Faculty of Medicine MU) and the Grant Agency of Masaryk University (MUNI/A/1565/2018).

This protocol was derived from the original research paper “Primary Mammary Organoid Model of Lactation and Involution” (Sumbal et al., 2020b).

Competing interests

The authors declare that they have no competing interests.

Ethics

The animal study was reviewed and approved by French legislation in compliance with European Communities Council Directives (A 75-15-01-3) and the regulations of the Institut Pasteur Animal Care Committees (CETEA).

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