A Robust Mammary Organoid System to Model Lactation and Involution-like Processes.

The mammary gland is a highly dynamic tissue that changes throughout reproductive life, including growth during puberty and repetitive cycles of pregnancy and involution. Mammary gland tumors represent the most common cancer diagnosed in women worldwide. Studying the regulatory mechanisms of mammary gland development is essential for understanding how dysregulation can lead to breast cancer initiation and progression. Three-dimensional (3D) mammary organoids offer many exciting possibilities for the study of tissue development and breast cancer. In the present protocol derived from Sumbal et al., we describe a straightforward 3D organoid system for the study of lactation and involution ex vivo. We use primary and passaged mouse mammary organoids stimulated with fibroblast growth factor 2 (FGF2) and prolactin to model the three cycles of mouse mammary gland lactation and involution processes. This 3D organoid model represents a valuable tool to study late postnatal mammary gland development and breast cancer, in particular postpartum-associated breast cancer. Graphic abstract: Mammary gland organoid isolation and culture procedures.

pregnancy, the mammary gland begins a new morphogenetic step initiated by hormonal stimulation, which is characterized by massive proliferation for epithelial expansion and alveolar development accompanied by adipocyte regression (Brisken and O'Malley, 2010). Importantly, prolactin signaling plays a crucial role in the terminal differentiation of luminal cells to enable milk production (Ormandy et al., 1997). At the end of lactation after weaning of the progeny, the mammary gland enters the Histologically, the mammary gland is composed of a bilayered epithelium consisting of an inner layer of luminal cells (keratin 8+) and an outer layer of contractile basal cells (keratin 5+). Luminal cells are responsible for milk production during lactation, while basal cells aid milk ejection. The epithelium is surrounded by a stromal fat pad that comprises fibroblasts, nerves, vasculature, lymphatics, immune cells, adipocytes, and extracellular matrix (ECM) (Richert et al., 2000).
Over the past decade, organoids of various tissues, such as stomach, colon, lung, and pancreas, have been developed (Huch and Koo, 2015), offering many exciting possibilities for the study of tissue development and disease. The organoid system is a powerful tool that combines the advantages of a Several models have been developed to study the mechanisms of mammary branching morphogenesis in primary mammary epithelium using different protocols (Ewald et al., 2008;Huebner et al., 2016;Neumann et al., 2018), cell lines (Xian et al., 2005), sorted cells (Jamieson et al., 2017;Linnemann et al., 2015), or induced pluripotent stem cells (Qu et al., 2017). However, an organoid system modeling key aspects of the late postnatal developmental stages of the mammary gland has remained challenging to establish.
Previously, there have been several attempts to model lactation in 3D culture: spheroids of a breast adenoma cell line were used to study copper secretion into milk (Freestone et al., 2014); organoids of primary epithelium were shown to produce milk following the administration of a lactogenic stimulus (Mroue et al., 2015;Jamieson et al., 2017); and co-culture of breast epithelium and pre-adipocyte cell lines was shown to initiate an involution-like process (Campbell et al., 2014). However, in-depth characterization of milk production and involution or the proper bilayered architecture of mammary epithelium remained to be carried out.
b. Sanitize the ventral side of the animal by spraying 70% EtOH on the skin. antibiotic supplements (gentamicin in digestion solution; penicillin and streptomycin in culture medium) will prevent the occurrence of contamination.
c. Pin the mouse by its four paws to a dissection board, with the abdomen facing upward (see Figure 1A, pins 1-4).
d. Using forceps, tightly grasp the skin of the lower part of the abdomen at half the width (see Figure 1A, point A). e. 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.
f. Continue to incise the skin cranially to the throat of the animal (see Figure 1A, from point A to point B).
g. 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).
h. 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).
i. Repeat step 8 on the other side of the animal (see Figure 1B, pins 8-10). The mammary glands are now exposed.
j. 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.
k. 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.
l. 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. m. Properly dispose of the animal corpse and continue with mechanical and enzymatic dissociation of the mammary glands.

Mechanical and enzymatic dissociation
Reminder: Work inside a laminar flow hood to maintain aseptic conditions. a. Freshly prepare 10 ml dissociation solution for the four glands collected from one mouse, pass through a 0.2-μm filter, and pre-heat at 37°C. c. Use three scalpels to finely chop the mammary glands and obtain a homogeneous mince of 1-mm 3 mammary fragments (see Figure 1C). d. Transfer the mince to a 50-ml tube containing the pre-warmed dissociation solution.
e. Place the tube in a shaking incubator for 30 min at 37°C, 100 rpm. Copyright  f. After incubation, resuspend the dissociated mammary glands by performing ten up-anddown motions with a 10-ml pipette. Centrifuge for 10 min at 400 × g.
g. After centrifugation, handle the 50-ml tube carefully to prevent disturbance of the three separated layers (see Figure 1C)  d. Count the organoids under the microscope at 4× magnification (see Figure 1D).

Notes:
i. Take each quarter of the cross as a landmark to avoid double-counting of the same organoid.
ii. 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) ii. 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). i. 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). j. In the meantime, pre-warm BOM at 37°C. k. Following incubation, carefully add 1 ml pre-heated BOM to each well and culture in the cell incubator at 37°C, 5% CO2.

Notes:
i. Add medium against the edges of the well to avoid disruption of the dome.
ii. 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).

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.  c. Renew all medium with BOM every two days, for a total of 8 days of treatment. versus d18), or at the morphological level by the progressive disappearance of branching (see Figure 2B and Figure 3B).   c. 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). d. 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. e. 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.
g. Incubate overnight at 65°C in a second bath of 100% melted paraffin.
h. Embed in a histology tissue mold using an embedding workstation.
i. Unmold the paraffin blocks after 24 h of solidification.
j. Cut 5-μm sections and spread on microscope slides. Keep the slides at room temperature until further analysis.

Dissociation solution
Note: This solution is prepared inside a laminar flow hood under aseptic conditions and does not need to be filter-sterilized.