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May 2020

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A Robust Mammary Organoid System to Model Lactation and Involution-like Processes
一个健康的乳腺有机样系统来模拟哺乳和退化样过程   

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Abstract

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


Keywords: Mouse (小鼠), Mammary gland (乳腺), 3D organoid (3D细胞器), Ex vivo (体外), Lactation (哺乳期), Involution (退化)

Background

The primary function of the mammary gland is to provide nutrition to newborns via milk production. The development of the mammary gland is a highly dynamic process that occurs mainly after birth and is regulated by several factors including hormones and growth factors (Brisken and Rajaram, 2006; Sternlicht, 2006). During puberty, hormones and growth factors regulate ductal morphogenesis from a rudimentary embryonic ductal tree (Brisken and O'Malley, 2010). During each 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 involution stage characterized by programmed cell death, tissue remodeling, and redifferentiation of adipocytes (Hughes and Watson, 2012; Macias and Hinck, 2012; Zwick et al., 2018; Jena et al., 2019).


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 2D culture (easy manipulation, precise control of cell composition and microenvironment, live imaging) with the opportunity to study complex cell–cell and cell–ECM interactions in a more controlled ex vivo manner (Huch and Koo, 2015; Shamir and Ewald, 2015; Koledova, 2017; Artegiani and Clevers, 2018).


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.


Recently, we developed a model of lactation and involution of mammary epithelium based on organoids of primary mammary gland tissue cultured in 3D Matrigel® (Sumbal et al., 2020b). Under lactogenic stimuli, primary organoids maintain long-term milk production, retain the contractile myoepithelial layer, and enter involution following hormone withdrawal. Moreover, after involution, the organoids remain hormonally sensitive and are able to enter another round of lactation (Sumbal et al., 2020b). Here, we present a methodological guideline to establish the primary mammary organoid-based ex vivo model of lactation and involution, with detailed procedures for obtaining tissue, isolating organoids, establishing and maintaining 3D culture, and preparing organoid samples for subsequent RNA or protein expression analysis or histological examination. This model can be used for studies on lactation biology, mammary stem cell plasticity, regulatory mechanisms of mammary epithelial cell differentiation and death, or other interesting biological phenomena. We believe that this model will initiate the further development of organoid technology, including creative applications in biotechnology and regenerative medicine (Sumbal et al., 2020a).


Materials and Reagents

  1. 100-mm tissue culture Petri dish (e.g., Corning, catalog number: 353003 )

  2. 0.2-μm filters and 50 ml syringes (e.g., GVS, catalog number: FJ25ASCCA002DL01 )

  3. No. 22 disposable scalpel blades (e.g., Swann-Morton, catalog number: 0508)

  4. 50-ml tubes (e.g., Corning, catalog number: 352070 )

  5. 15-ml tubes (e.g., Corning, catalog number: 352096 )

  6. 10-ml disposable plastic pipettes (e.g., Corning, catalog number: 357551 )

  7. 25-ml disposable plastic pipettes (e.g., Corning, catalog number: 357535 )

  8. 24-well tissue culture plates (e.g., Corning, catalog number: 353047 )

  9. 30 G insulin syringes (e.g., BD Microfine, catalog number: 324826 )

  10. Plastic histology molds (e.g., Thermo Scientific, catalog number: 1830 )

  11. Plastic embedding cassettes (e.g., Simport, catalog number: M492-2 )

  12. Histology tissue molds (e.g., Simport, catalog number: M474-3 )

  13. Microscope slides for histology (e.g., Thermo Scientific, catalog number: J1800AMNZ )

  14. Mice: virgin females, 7–10 weeks old, inbred strain C57BL/6J (e.g., The Jackson Laboratory, catalog number: 000 664 )

  15. Ethanol (EtOH), 70%, 95%, and 100% (e.g., VWR, catalog number: 83813 )

  16. Phosphate-buffered saline (PBS) (e.g., Sigma-Aldrich, catalog number: D1408 )

  17. Dulbecco’s modified Eagle medium (DMEM)/F12 (e.g., Gibco, catalog number: 21331-020 )

  18. Bovine serum albumin (BSA) (e.g., Sigma-Aldrich, catalog number: A3608 )

  19. Fetal bovine serum (FBS) (e.g., Sigma-Aldrich, catalog number: F0804 )

  20. Collagenase A (e.g., Roche, catalog number: 11088793001 )

  21. Trypsin (e.g., Dutcher Dominique, catalog number: P10-022100 )

  22. Insulin (e.g., Sigma-Aldrich, catalog number: I6634-100MG )

  23. Gentamicin (e.g., Sigma-Aldrich, catalog number: G1397 )

  24. Glutamine (e.g., Gibco, catalog number: 35050-061 )

  25. DNase I (e.g., Sigma-Aldrich, catalog number: D4527-40KU )

  26. Dispase II (e.g., Roche, catalog number: 13 75 2000 )

  27. Growth factor-reduced Matrigel® (e.g., Corning, catalog number: 354230 )

  28. Insulin-transferrin-selenium (ITS) (e.g., Gibco, catalog number: 41400-045 )

  29. Penicillin/Streptomycin (e.g., Gibco, catalog number: 15140-122 )

  30. FGF2 (e.g., Gibco, catalog number: PM60034 )

  31. Prolactin (e.g., Sigma-Aldrich, catalog number: SRP4688 )

  32. Hydrocortisone (e.g., Sigma-Aldrich, catalog number: S H6909 )

  33. Oxytocin (e.g., Sigma-Aldrich, catalog number: O3251 )

  34. RNeasy Micro Kit (e.g., Qiagen, catalog number: 74004 )

  35. β-Mercaptoethanol (e.g., Sigma-Aldrich, catalog number: M6250 )

  36. Phosphatase inhibitor cocktail II (e.g., Millipore, catalog number: 524625 )

  37. RIPA buffer (e.g., Sigma-Aldrich, catalog number: R0278 )

  38. Protease inhibitor cocktail I (e.g., Sigma-Aldrich, catalog number: 539131 )

  39. Pierce Coomassie (Bradford) Protein Assay Kit (e.g., Thermo Scientific, catalog number: 23200 )

  40. Paraformaldehyde (PFA), 32% (e.g., Electron Microscopy Sciences, catalog number: 15714 )

  41. Low gelling temperature agarose (e.g., Sigma-Aldrich, catalog number: A9414 )

  42. Xylene (e.g., Sigma-Aldrich, catalog number: 534056 )

  43. Paraffin (e.g., Sigma-Aldrich, catalog number: 1071642504 )

  44. Dissociation solution (see Recipes)

  45. BSA solution (see Recipes)

  46. Basal organoid medium (BOM) (see Recipes)

  47. Morphogenesis medium (see Recipes)

  48. Lactation medium (see Recipes)

  49. 4% PFA (see Recipes)

  50. RNA lysis buffer (see Recipes)

Equipment

  1. Surgical tools

    Forceps (e.g., Phymep, catalog numbers: 11050-10 and 11051-10 )

    Scissors (e.g., Phymep, catalog number: 14088-10 )

  2. Dissection board (e.g., Thermo Scientific, catalog number: 36-119 )

  3. P1000 pipette

  4. Laminar flow hood

  5. Fridge 4°C (e.g., Liebherr, catalog number: 7083 001-01 )

  6. Freezer –80 °C (e.g., Thermo Scientific, catalog number: 88400V )

  7. Liquid nitrogen tank (e.g., Air Liquide Espace 151, catalog number: 2433867 )

  8. Shaking incubator at 37°C (e.g., Infors HT Multitron)

  9. Centrifuge (e.g., Thermo Scientific, model: Sorvall ST40 )

  10. Incubator for cell culture, 37°C, 5% CO2 (e.g., Thermo Scientific, model: HERAcell 150i )

  11. Heating plate at 37°C (e.g., Techne DRI-Block DB-2A)

  12. Microscope and camera (e.g., Olympus model: CKX41 )

  13. NanoDrop™ (e.g., Implen Nanophotomoter NP80)

  14. Sonicator (e.g., Diagenode Bioruptor Pico)

  15. Incubator at 65°C (e.g., Memmert Incubator I)

  16. Embedding workstation (e.g., Leica EG1150C )

Procedure

  1. 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

    3. Reminder: Work inside a laminar flow hood to maintain aseptic conditions.

      1. 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.

        Note: Do not exceed the maximum 30 ml dissociation solution in a 50-ml tube to ensure correct dissociation.

      2. Transfer the freshly collected mammary glands to a new sterile Petri dish.

      3. Use three scalpels to finely chop the mammary glands and obtain a homogeneous mince of 1-mm3 mammary fragments (see Figure 1C).

      4. Transfer the mince to a 50-ml tube containing the pre-warmed dissociation solution.

      5. Place the tube in a shaking incubator for 30 min at 37°C, 100 rpm.

        Notes:

        1. All the following steps are performed at room temperature except incubation with dispase.

        2. From here on, pre-coat all the pipettes, tips, and tubes with 2.5% BSA solution. Prepare the BSA solution in a 50-ml tube and aspirate/remove from every consumable following coating; this will prevent stickiness and loss of organoids. The BSA solution can then be filtered, stored at 4°C, and re-used.

      6. After incubation, resuspend the dissociated mammary glands by performing ten up-and-down motions with a 10-ml pipette. Centrifuge for 10 min at 400 × g.

      7. After centrifugation, handle the 50-ml tube carefully to prevent disturbance of the three separated layers (see Figure 1C). Keep the epithelial pellet intact and transfer the middle aqueous phase and the top fatty layer into a clean 15-ml tube.

      8. Resuspend the epithelial pellet in 5 ml DMEM/F12 and set it aside.

      9. Focus on the fatty and aqueous solutions in the 15-ml tube: resuspend by performing ten up-and-down motions with a 10-ml pipette. Centrifuge for 10 min at 400 × g.

        Note: This step allows recovery of epithelial fragments trapped in the fatty layer.

      10. Again, handle the 15-ml tube carefully to avoid disturbing the three separated layers. Discard the fatty and aqueous layers.

      11. Take the 5 ml resuspended pellet from the 50-ml tube to resuspend the pellet in the 15-ml tube.

      12. Wash the 50-ml tube with 5 ml DMEM/F12, pool with the suspension in the 15-ml tube, and mix.

      13. Centrifuge for 10 min at 400 × g.

      14. Discard the supernatant. Use 4 ml DMEM/F12 to resuspend the pellet. Subsequently, add 80 μl DNAse I at 100 μg/ml and agitate for 5 min by hand or on an orbital shaker at 100 rpm.

      15. Add 6 ml DMEM/F12 and resuspend the solution by performing 5 up-and-down motions with a 10-ml pipette.

      16. Centrifuge for 10 min at 400 × g.

      17. Discard the supernatant. Use 4 ml DMEM/F12 to resuspend the pellet. Subsequently, add 150 μl dispase II at 0.5 mg/ml and incubate for 5 min at 37°C.

      18. Add 6 ml DMEM/F12 and resuspend the solution by performing 5 up-and-down motions with a 10-ml pipette.

      19. Centrifuge for 10 min at 400 × g.

      20. Discard the supernatant. Resuspend the pellet in 9 ml DMEM/F12.

      21. Perform differential centrifugation to separate the mammary epithelium from the stromal fraction: centrifuge the suspension for 15 s at room temperature, 400 × g. Discard the supernatant containing the stromal fraction and resuspend the epithelial pellet in 9 ml DMEM/F12.

        Note: Set the time on the centrifuge to 1 min. Once a speed of 400 × g is reached, time 15 s precisely and stop the centrifuge manually.

      22. Repeat the previous step (t) 4 times, for a total of 5 differential centrifugations, to efficiently remove stromal contamination.

      23. Resuspend the final pellet in 1 ml basal organoid medium (BOM) and place on ice. The organoids are now ready to be counted and cultured.

        Note: Adjust the volume of resuspension according to pellet size. From a pool of 2–3 mice, the final pellet was resuspended in 1 ml basal organoid medium, for an expected range of 3,000–6,000 organoids. Adjust the volume of BOM for resuspension of the pellet according to the number of mice pooled.



      Figure 1. Key steps of mammary gland collection for organoid isolation and 3D culture. A,B. Images of mouse dissection to access the mammary gland. A. Needles 1–4 represent the points at which to pin the mouse. Needles 5–7 and 8–10 represent the points at which to pin the skin of the mouse. Letters A–F with the blue dotted lines indicate the cuts. B. Green dotted lines denote the mammary gland. The lymph node is denoted in red and must be removed. C. Mammary gland before (left panel) and after (middle panel) mincing with a scalpel. Mammary organoids after transfer to dissociation medium (right panel). D. Example of mammary organoid counting. Left panel: organoids are surrounded by dotted lines. Star represents nerves. Right panel: organoids after embedding in Matrigel®. Arrow represents the edge of the Matrigel® dome. Scale bar = 500 μm. E. Freshly isolated primary organoid. Left panel: image of a mammary organoid on day 1 post-isolation. Middle panel: Hematoxylin & eosin staining of an organoid on day 1 post-isolation. Right panel: immunofluorescence staining showing the distribution of myoepithelial (keratin 5+, green) and luminal cells (keratin 8+, red) in organoids on day 1 post-isolation. Hoechst, blue (nuclei). Scale bar = 100 μm.

    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.


  1. 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

  1. 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

  2. BSA solution

    Note: This solution can be filter-sterilized and reused several times when stored at 4°C.

    2.5% BSA in PBS

  3. 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

  4. 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

  5. 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

  6. 4% PFA

    Note: This solution is prepared inside a chemical hood and does not need to be filter-sterilized.

    4% paraformaldehyde in PBS

  7. 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).

  8. 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

  9. 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).

References

  1. Artegiani, B., and Clevers, H. (2018). Use and application of 3D-organoid technology. Hum Mol Genet 27: R99-R107.
  2. Brisken, C. and O'Malley, B. (2010). Hormone action in the mammary gland. Cold Spring Harb Perspect Biol 2(12): a003178.
  3. Brisken, C. and Rajaram, R. D. (2006). Alveolar and lactogenic differentiation. J Mammary Gland Biol Neoplasia 11(3-4): 239-248.
  4. Campbell, J. J., Botos, L. A., Sargeant, T. J., Davidenko, N., Cameron, R. E. and Watson, C. J. (2014). A 3-D in vitro co-culture model of mammary gland involution. Integr Biol (Camb) 6(6): 618-626.
  5. Ewald, A. J., Brenot, A., Duong, M., Chan, B. S. and Werb, Z. (2008). Collective epithelial migration and cell rearrangements drive mammary branching morphogenesis. Dev Cell 14(4): 570-581.
  6. Freestone, D., Cater, M. A., Ackland, M. L., Paterson, D., Howard, D. L., de Jonge, M. D. and Michalczyk, A. (2014). Copper and lactational hormones influence the CTR1 copper transporter in PMC42-LA mammary epithelial cell culture models. J Nutr Biochem 25(4): 377-387.
  7. Huch, M. and Koo, B. K. (2015). Modeling mouse and human development using organoid cultures. Development 142(18): 3113-3125.
  8. Huebner, R. J., Neumann, N. M. and Ewald, A. J. (2016). Mammary epithelial tubes elongate through MAPK-dependent coordination of cell migration.Development 143: 983-993.
  9. Hughes, K. and Watson, C. J. (2012). The spectrum of STAT functions in mammary gland development. JAKSTAT 1(3): 151-158.
  10. Jamieson, P. R., Dekkers, J. F., Rios, A. C., Fu, N. Y., Lindeman, G. J. and Visvader, J. E. (2017). Derivation of a robust mouse mammary organoid system for studying tissue dynamics.Development 144(6): 1065-1071.
  11. Jena, M. K., Jaswal, S., Kumar, S. and Mohanty, A. K. (2019). Molecular mechanism of mammary gland involution: An update. Dev Biol 445(2): 145-155.
  12. Koledova, Z. (2017). 3D Cell Culture: An Introduction. Methods Mol Biol 1612: 1-11.
  13. Linnemann, J. R., Miura, H., Meixner, L. K., Irmler, M., Kloos, U. J., Hirschi, B., Bartsch, H. S., Sass, S., Beckers, J., Theis, F. J., Gabka, C., Sotlar, K. and Scheel, C. H. (2015). Quantification of regenerative potential in primary human mammary epithelial cells. Development 142(18): 3239-3251.
  14. Macias, H. and Hinck, L. (2012). Mammary gland development. Wiley Interdiscip Rev Dev Biol 1(4): 533-557.
  15. Mroue, R., Inman, J., Mott, J., Budunova, I., and Bissell, M.J. (2015). Asymmetric expression of connexins between luminal epithelial- and myoepithelial- cells is essential for contractile function of the mammary gland.Dev Biol 399(1): 15-26.
  16. Neumann, N. M., Perrone, M. C., Veldhuis, J. H., Huebner, R. J., Zhan, H., Devreotes, P. N., Brodland, G. W. and Ewald, A. J. (2018). Coordination of Receptor Tyrosine Kinase Signaling and Interfacial Tension Dynamics Drives Radial Intercalation and Tube Elongation. Dev Cell 45(1): 67-82 e66.
  17. Ormandy, C. J., Camus, A., Barra, J., Damotte, D., Lucas, B., Buteau, H., Edery, M., Brousse, N., Babinet, C., Binart, N. and Kelly, P. A. (1997). Null mutation of the prolactin receptor gene produces multiple reproductive defects in the mouse. Genes Dev 11(2): 167-178.
  18. Qu, Y., Han, B., Gao, B., Bose, S., Gong, Y., Wawrowsky, K., Giuliano, A. E., Sareen, D. and Cui, X. (2017). Differentiation of Human Induced Pluripotent Stem Cells to Mammary-like Organoids. Stem Cell Reports 8(2): 205-215.
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  22. Sumbal, J., Budkova, Z., Traustadottir, G. A. and Koledova, Z. (2020a). Mammary Organoids and 3D Cell Cultures: Old Dogs with New Tricks. J Mammary Gland Biol Neoplasia. doi: 10.1007/s10911-020-09468-x.
  23. Sumbal, J., Chiche, A., Charifou, E., Koledova, Z. and Li, H. (2020b). Primary Mammary Organoid Model of Lactation and Involution. Front Cell Dev Biol 8: 68.
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简介


[摘要]乳腺是一种高度动态的组织,在整个生殖生活中都会发生变化,包括青春期的生长以及怀孕和进化的重复周期。乳腺肿瘤诊断代表在世界女性最常见的癌症宽。研究的监管机制乳腺的发育是至关重要的理解荷兰国际集团d如何YS调节可导致乳腺癌的发生和发展。三维(3D)乳腺组织体提供了许多令人激动的可能性的研究的组织发育和乳腺癌。在第E存在衍生自协议Sumbal等人,我们描述一个简单的3D类器官系统的研究的泌乳和复古体外。我们使用成纤维细胞生长因子2 (FGF2)和催乳素刺激的原代和传代小鼠乳腺类器官来模拟小鼠乳腺泌乳和内卷过程的三个周期。这种3D模型类器官代表一个有价值的工具来研究后期产后乳腺的发育和乳腺癌,尤其是产后-相关性乳腺癌。


图形摘要:


乳腺类器官的分离和培养程序

[背景技术]的Th e是乳腺的主要功能是提供营养吨经由牛奶产量Ò新生儿。牛逼乳腺他的发展是主要发生在出生后,由几个因素,包括激素和生长因子调控的一个高度动态的过程(Brisken和拉贾拉姆,2006;斯特恩利希特,2006年)。在青春期,激素和生长因子调节基本的胚胎导管树的导管形态发生(Brisken and O'Malley,2010)。在每次怀孕期间,乳腺开始由激素刺激启动的新的形态发生步骤,其特征在于上皮扩张和肺泡发育的大量增殖,伴随着脂肪细胞的退化(Brisken and O'Malley,2010)。重要的是,催乳素信号传导在腔细胞的终末分化中起着至关重要的作用,以使得能够产生乳汁(Ormandy等,1997)。子代断奶后的哺乳结束时,乳腺进入以细胞程序性死亡,组织重塑和脂肪细胞再分化为特征的对合阶段(Hughes和Watson,2012; Macias和Hinck,2012; Zwick等,2018)。; Jena et al。,2019 )。

在组织学上,乳腺由双层上皮组成,该双层上皮由腔细胞的内层(角蛋白8+)和收缩性基底细胞的外层(角蛋白5+)组成。泌乳过程中,发光细胞负责产奶,而基础细胞则有助于泌乳。的上皮被包围一个基质脂肪垫的是包括成纤维细胞,神经,脉管系统,淋巴管,免疫细胞,脂肪细胞,和细胞外基质(ECM) (里克特等人,2000) 。

在过去的十年中,类器官的各种组织,如胃,结肠,肺,胰腺,已经开发(胡赫与晟,2015年),提供了许多令人激动的可能性的研究的组织发育和疾病。邻rganoid系统是一个功能强大的工具,它结合了一个2D培养的与优点(易操纵,细胞组合物和微环境的精确控制,实时成像)的机会来研究复杂的细胞-细胞和细胞-在一个更控制研究-ECM相互作用编离体方式(Huch and Koo,2015; Shamir and Ewald,2015 ; Koledova,2017; Artegiani and Clevers,2018 )。

几个模型已经发展到研究的乳腺分支形态发生的机制在初级乳腺上皮细胞使用不同协议(Ewald的等人,2008;许布纳等人,2016;纽曼等人,2018) ,细胞系(冼等。,2005) ,分选的细胞(贾米森等人,2017 ; Linnemann 。等人,2015 ),或诱导的多能干细胞(曲等人,2017) 。然而,模拟乳腺产后晚期发育阶段的关键方面的类器官系统仍然难以建立。

以前,已经进行了多种尝试来模拟3D培养中的泌乳:乳腺腺瘤细胞系的球状体用于研究铜向乳汁中的分泌(Freestone等,2014)。类器官的初级上皮显示出生产牛奶以下一个的施用生乳的刺激(Mroue等人,2015 ;贾米森等人,2017年); 以及乳腺癌上皮细胞和前脂肪细胞系的共培养显示启动了内卷样过程(Campbell et al。,2014)。然而,对牛奶生产和复合或乳腺上皮的适当双层结构的深入表征仍有待进行。

最近,瓦特È开发泌乳和乳腺上皮细胞的退化的基于类器官的模型的三维培养的原代乳腺组织的Matrigel ® (Sumbal等人,2020B) 。在产乳刺激下,主要类器官可维持长期的产奶量,保留收缩的肌上皮层,并在激素撤除后进入内卷。此外,复性后,类器官仍然对激素敏感,并且能够进入另一轮泌乳期(Sumbal等人,2020b)。在这里,我们提出了一个方法指针以建立原代乳腺类器官的基于体外哺乳期和的模型乘方,与细节编程序用于获得组织,分离类器官小号,建立荷兰国际集团和MAINT癌宁3D培养,并prepar荷兰国际集团用于随后的器官样的样品RNA或蛋白表达分析或组织学检查。该模型可用于研究Ø ñ哺乳生物学,乳腺干细胞的可塑性,乳腺上皮细胞分化的调控机制和死亡,或其他有趣的生物现象。我们相信,该模型将启动类器官技术的进一步发展,包括在生物技术和再生医学中的创新应用(Sumbal等人,2020a)。

关键字:小鼠, 乳腺, 3D细胞器, 体外, 哺乳期, 退化

材料和试剂

100毫米的文化培养皿(例如,Corning,目录号:353003)
0.2 -微米˚F ilters一个ND 50个升注射器(例如,GVS,目录号:FJ25ASCCA002DL01)
Ñ O操作。22 d ISP奥萨卜莱手术刀片(例如,斯旺-莫顿,目录号:0508)
50 -毫升(管例如,康宁,目录号:352070)
15 -毫升管(例如,康宁,目录号:352096)
10 -毫升d isposable塑料移液管(例如,康宁,目录号:357551)
25 -毫升d isposable塑料移液管(例如,康宁,目录号:357535)
24孔吨问题培养板(例如,Corning公司,目录号:353047)
30 ģ我nsulin小号yringes(例如,BD微细,目录号:324826)
塑料组织学模具(例如,Thermo Scientific,目录号:1830)
塑料包埋盒(例如,Simport ,目录号:M492-2)
组织学组织模具(例如,Simport ,目录号:M474-3)
用于组织学的显微镜载玻片(例如,Thermo Scientific,目录号:J1800AMNZ)
小鼠:v irgin雌性,7 - 10周龄,近交系C57BL / 6J(例如,Jackson实验室,目录号:000664)
乙醇(EtOH中),70%,95% ,和100%(例如,VWR,目录号:83813)
磷酸盐-b uffer ED小号艾琳(PBS) (例如,Sigma-Aldrich公司,目录号:D1408)
Dulbecco氏米odified鹰米edium(DMEM)/ F12(例如,Gibco公司,目录号:21331-020)
牛小号erum一个lbumin(BSA)(例如,Sigma-Aldrich公司,目录号:A3608)
胎b绵羊小号erum(FBS)(例如,。Sigma-Aldrich公司,目录号:F0804)
胶原酶A(例如,Roche,目录号:11088793001)
胰蛋白酶(例如,Dutcher Dominique,目录号:P10-022100)
胰岛素(例如,Sigma-Aldrich公司,目录号:I6634-100MG)
庆大霉素(例如,Sigma-Aldrich公司,目录号:G1397)
谷氨酰胺(例如,Gibco公司,目录号:35050-061)
DNA酶I(例如,Sigma-Aldrich公司,目录号:D4527-40KU)
分散酶II(例如,罗氏,目录号:13 75 2000)
增长˚F演员-r得出基质胶® (例如,康宁公司,目录号:354230)
胰岛素吨ransferrin-小号elenium(ITS) (例如,Gibco公司,目录号:41400-045)
青霉素/链霉素(例如,Gibco公司,目录号:15140-122)
FGF2 (例如,Gibco公司,目录号:PM60034)
催乳素(例如,Sigma-Aldrich ,目录号:SRP4688)
氢化可的松(例如,Sigma-Aldrich公司,目录号:小号H6909)
催产素(例如,Sigma-Aldrich公司,目录号:O3251)
的RNeasy Micro试剂盒(例如,Qiagen公司,目录号:74004)
β-中号ercaptoethanol (例如,Sigma-Aldrich公司,目录号:M6250 )
磷酸酶抑制剂混合物II(例如,Millipore公司,目录号:524625)
RIPA缓冲液(例如,Sigma-Aldrich公司,目录号:R0278)
蛋白酶抑制剂混合物I(例如,Sigma-Aldrich公司,目录号:539131)
皮尔斯考马斯(布拉德福德)P rotein甲SSAY ķ它(例如,热科学,目录号:23200)
32%的多聚甲醛(PFA)(例如,电子显微镜科学,目录号:15714)
低胶凝温度的琼脂糖(例如,Sigma-Aldrich,目录号:A9414)
二甲苯(例如,Sigma-Aldrich公司,目录号:534056)
石蜡(例如,Sigma-Aldrich公司,目录号:1071642504)
解离解决方案(请参阅食谱)
BSA解决方案(请参阅食谱)
基础ø rganoid米edium(BOM)(见配方)
形态发生培养基(请参见食谱)
泌乳培养基(请参见食谱)
PFA为4%(请参阅食谱)
RNA裂解缓冲液(请参阅食谱)


设备


手术工具
镊子(例如,Phymep ,目录号:11050-10和11051-10)


剪刀(例如,Phymep ,目录号:14088-10)


解剖板(例如,Thermo Scientific,目录号:36-119)
P1000移液器
层流罩
冰箱4°C(例如,利勃海尔(Liebherr),目录号:7083 001-01)
冰柜– 80°C (例如,Thermo Scientific,目录号:88400V)
液氮罐(例如,Air Liquide Espace 151,目录号:2433867)
在37°C下摇动培养箱(例如,Infors HT Multitron )
离心机(例如,热电三英tific ,型号:SORVALL ST40)
用于细胞培养的培养箱,37°C,5%CO 2 (例如,Thermo Scientific ,型号:HERAcell 150i)
在37℃下加热板(例如,Techne公司DRI-块DB-2A)
显微镜和照相机(例如,奥林巴斯型号:CKX41)
纳米d ROP ™ (例如,Implen Nanophotomoter NP80)
超声仪(例如,Diagenode Bioruptor Pico)
培养箱中在65℃(例如,Memmert培养箱I)
嵌入工作站(例如,Leica EG1150C)


程序


乳腺主要类器官的分离
处女亩的夹层本身收获乳腺(SE ë V记意1)。




视频1.收集乳腺。该视频是在巴斯德研究所制作的。根据从指引的规定研究所巴斯德动物保护委员会(CETEA)。。在动物保健的批准,符合欧共体理事会指令(A 75-15-01-3法国立法)。


使用符合道德规范的方法(例如,颈脱位)安乐死供体小鼠,并立即进行乳腺收集。
注意小号:


颈椎脱位是一种常见的方法对动物实施安乐死,并提供š快速,无痛的死亡。使用这种方法,如果立即收集,则不会改变培养物中细胞/组织的存活率。
在处理多个小鼠的情况下,一个安乐死动物和搜集的腺体立即,然后继续到下一个动物。
通过在皮肤上喷洒70%的乙醇对动物的腹侧进行消毒。
注意:消毒后,请在层流通风橱内工作以保持无菌状态。的无菌工作程序的应用程序,与存在一起抗真菌剂和抗生素补充(在消化溶液庆大霉素;青霉素和链霉素中培养MEDI微米)将防止污染的发生。


销通过它的四只爪子鼠标到夹层板,与腹部朝上病房(见图1A,销1 - 4)。
使用镊子,紧紧GRA用上皮肤ø ˚F一半的宽度腹部的下部(参见图1A,点A)。
用手术剪,使第一切口我ñ在A点的皮肤
注意:小心只切开皮肤,不要使下面的腹膜破裂。


继续从动物的喉咙开颅切开皮肤(参见图1A,从A点到B点)。
从该中间线,使用镊子GRA SP皮肤和切口朝向四个爪的(参见图1A,用切口加入所述中间线来分C,d,E或F ,分别)。
用镊子和棉签轻轻地将动物的一侧的皮肤与腹膜分开。用三个销钉将皮肤连接到解剖板上(请参见图1B,销钉5 – 7)。
对动物的另一侧重复步骤8(见图1B,销8 - 10)。第m ammary腺现在露出。
鉴定乳腺的淋巴结腺#4(一个小的致密结构,圆在形状;参见图1B,包围)。ř EMOVE淋巴结使用镊子和剪刀和废弃都腺体。
继续收获#3和#4乳腺。使用弯钳,克锉刀乳腺,轻轻地分隔符从皮肤和其他组织机智Ë他们ħ剪刀。
注:仔细分离乳腺#3(发白和光泽)从肌肉(浅棕色棱纹的结构),因为该协议并没有防止肌肉污染。


P花边在相同的无菌皮氏培养皿含有冷PBS所有收集的腺体(一个pproximately 3 ml的,先前存储在4℃下)为洗荷兰国际集团之前组织处理。
正确处置动物尸体,并继续进行乳腺的机械和酶解。
机械和酶解离
提醒:在层流罩内工作以保持无菌状态。


新鲜制备出10 ml的解离溶液的四个压盖小号从一只小鼠收集,传递通过0.2 - μ在37微米的过滤器,并预热℃。
注意:不要超过最大30毫升中分离溶液一个50 -毫升管,以确保正确解离。


将新鲜收集的乳腺转移到新的无菌培养皿中。
用三个手术刀将乳腺切碎,并均匀切碎1 - mm 3的乳腺碎片(见图1C)。
转移剁碎到50 -毫升包含预温热解离溶液管。
放置管我Ñ在37℃,100rpm下进行30分钟的振荡培养箱中。
笔记:


除与分散酶孵育外,以下所有步骤均在室温下进行。
从这里开始,用2.5%BSA溶液预涂所有移液器,吸头和试管。制备BSA溶液,在50 -毫升管和抽吸/删除从每消耗品以下涂层; Ť他将防止粘性和组织体的损失。所述然后BSA溶液可以被过滤,储存在4℃下,并重新使用。
温育后,重悬由离解的乳腺进行10向上和向下运动,用10 -毫升移液管。以400 × g离心10分钟。
甲压脚提升离心,手柄50 -毫升管小心,以防止三个分离层的干扰(浏览F igure 1C)。保持上皮沉淀完整和中间水相和顶部脂肪层转移到干净的15 -毫升管中。
重悬沉淀上皮5 ml的DMEM / F12和设置它放在一边。
着眼于脂肪和水溶液š在15 -毫升:管再悬浮通过执行10向上和向下运动,用10 -毫升移液管TE。以400 × g离心10分钟。
注意:该步骤允许恢复Y的EP ithelial片段被困在脂肪层。


再次,把手15 -毫升管小心以避免扰乱三个分离层。丢弃脂肪层和水层。
采取第E(5)毫升ř esuspended粒料从50 -毫升管以重悬浮在15沉淀-毫升管中。
洗50 -毫升用5管毫升DMEM / F12 ,池在15悬浮液-毫升管,并混合。
以400 × g离心10分钟。
丢弃上清液。用4 ml DMEM / F12重悬沉淀。随后,加入80微升DNA酶在100 I微克/毫升并搅拌5分钟通过手或上以100rpm的轨道摇床。
加6毫升DMEM / F12和重悬由溶液进行5向上和向下运动,用10 -毫升移液管。
以400 × g离心10分钟。
丢弃上清液。使用4 ml DMEM / F12重悬沉淀。接着,添加150 μl的分散酶以0.5mg / II毫升孵育为5分钟,在37℃。
加6毫升DMEM / F12和重悬由溶液进行5向上和向下运动,用10 -毫升移液管。
以400 × g离心10分钟。
丢弃上清液。将沉淀重悬于9 ml DMEM / F12中。
执行差异离心分离的乳腺上皮细胞从基质部分:Ç entrifuge悬浮液15秒,在室温下,400 ×克。丢弃含有上清液的基质部分,重悬沉淀上皮9 ml的DMEM / F12。
注意:将离心机上的时间设置为1分钟。一旦达到400 × g的速度,则精确计时15 s,并手动停止离心机。


重复上一步骤(t)4次,总共进行5次差异离心,以有效去除基质污染。
将最终沉淀重悬于1 ml基础类器官介质(BOM)中,并置于冰上。邻rganoids是现在准备要凑nted和培养。
注意:甲djust重悬浮的体积根据粒料尺寸。从2的池- 3只小鼠,将最终的沉淀重悬于1 ml的基肥类器官培养基,为3的预期范围,000 - 6 ,000类器官。调整BOM的体积为resuspen的锡永根据汇集的小鼠的数目沉淀。




图1.用于类器官分离和3D培养的乳腺采集的关键步骤。A ,B 。图片鼠标清扫访问牛逼他乳腺。A.针1 - 4代表点š处到引脚鼠标。针5 -图7和8 - 10代表点小号处到引脚的小鼠的皮肤。带蓝色虚线的字母A – F表示切口。B. ģ颖虚线表示的乳腺。淋巴结用红色表示,必须将其切除。C.乳腺前(左面板),并用切碎后(中图)腺一个手术刀。转让后乳腺组织体以解离MEDI微米(右图)。D.乳腺类器官计数的例子。左侧面板:直径:rganoids包围由虚线表示。星代表神经。右图:Ø在嵌入后rganoids中号ATRIGEL ® 。箭头表示的边缘的中号ATRIGEL ®圆顶。比例尺= 500 μ米。E.新鲜分离的主要类器官。左侧面板:图像乳腺组织体上每天1后的隔离。中图:苏木和伊红染色的一类器官在第1天后隔离。右图:我mmunofluorescen CE染色显示出荷兰国际集团的肌上皮(角蛋白5的分布+ ,绿色)和管腔细胞(角蛋白8 +在类器官,红色)上天1后的隔离。赫斯特,蓝色(原子核)。比例尺= 100 μ米。


类器官计数
提醒:在层流罩内工作以保持无菌状态。


d生两个大杂交用标记ö Ñ显微镜载玻片。
采取类器官悬浮液,并使用P 1000移液器执行五次上下运动以使其均质化。
在玻片的背面,在每个十字的中心周围散布10μl溶液。
注:使用20 -微升尖或切10的末端-微升提示,以避免大的组织体进行来临被困。


算机关ø IDSù的nDer显微镜在4 ×放大倍数(见图1D)。
注意小号:


以交叉的每个季度的一个里程碑,以避免重复-同一个组织体计数。
类器官呈圆形结构,周边光滑。偶尔和事无巨细,神经和血管内皮细胞都还存在。神经小号出现如绳索状结构,并且可以在束被组织(参见图1D)。内皮有一个比较有点衣衫褴褛的外观与光滑的外观类器官。初级神经和内皮细胞的少量存在不会干扰类器官的泌乳或退化。  
只计算了一个直径类器官伟大超过30 ER - 50微米,因为在较小的中号AY不能正常发育。
计算平均值两者的计数在10微升溶液中,乘法根据VOLU BOM的我用于重悬沉淀,以获得所述类器官的总数。
注意:将新鲜分离的有机anoids可以在FBS中的溶液被可行地冻结含10%DMSO的长-在LIQ长期储存UID NITR蛋白原和以后使用。


B.乳腺类器官的3D培养     

嵌入在基质胶®
提醒:在层流罩内工作以保持无菌状态。洗冰桶和加热板用70%彻底EtOH中之前PLAC EMENT在层流罩。


解冻的基质胶®在冰上或4℃。
笔记:


基质胶®凝固的真快于室温。在使用前和电镀过程中,请始终将其放在冰上。
请记住,基质胶®解冻需要时间; 吨herefore,开始解冻之前的步骤(对于1 2小时-毫升等分试样,对于10 6小时-毫升瓶)。
将24孔板放在冰上。计算所需井和蔓延20号-微升基质胶在每个孔的底部一个圆形贴片® 。
注:开始通过将尖端含基质胶®在井的中心并朝向井的边缘圆扩大,而不触及他们。


在细胞培养箱(5%CO 2 )中于37°C孵育24孔板15分钟。
同时,将加热板预热到37°C 。
制备的中类器官悬浮的基质胶® :Ç alculate类器官悬浮液的体积需要获得的类器官的期望数量。吸管这个体积悬浮液的到新鲜1.5 -毫升管中并离心3分钟,在400 ×克。
注意:调节类器官的每取决于实验的类型以及数量:每孔200类器官用于形态学和组织学,400为基因表达,和1000瓦特西部时代印迹廷分析。


小心除去上清液,然后将试管放在冰上。随后,小心地重新悬浮在冷的所需体积颗粒基质胶® (50微升每孔),避免气泡形成。保持冰上。
从细胞培养箱中取出24孔板,放在37°C的加热板上。
在每一个基质胶® -预涂井,谨慎种子类器官的悬浮液(在基质胶®)作为一个拱顶之上的固化的基质胶®补丁。
放置24孔板回到细胞培养箱(5%CO 2 )30分钟,在37℃以固化该基质胶® (见˚F igure 1D)。
同时,在37°C下预热BOM。
以下温育后,小心地加入1毫升预热BOM至在37℃,5%CO每个孔中并培养在细胞培养箱2 。
注意小号:


添加mediu中号对井的边缘的圆顶避免影响。
可以执行类器官的表征使用常规的组织学染色剂(例如,苏木精和曙红)或免疫染色在一天1后在BOM恢复(参见图1E和步骤的程序的B2) 。
FGF2的形态发生
提醒:在层流罩内工作以保持无菌状态。


注意:过量回收对于类器官培养是最佳的;然而,FGF2治疗可以施用立即电镀后的Ò rganoids。


将BOM预先加热到37°C。
添加新鲜FGF2在一个终浓度的2.5 nM的至预加热的BOM ,得到形态发生平台。
从吸孔中的介质不接触的Matrigel ®圆顶,并用800替换微升新鲜形态平台。
每3天用新鲜的形态发生培养基更新所有培养基,共6天。
催乳素的致乳剂分化
将BOM预先加热到37°C。
将1μg / ml催乳素和1μg / ml氢化可的松添加到预热的BOM中以获得泌乳培养基。
从吸孔中的介质不接触的Matrigel ®圆顶,并用800替换微升新鲜泌乳平台。
每两天用新鲜的泌乳培养基更新所有培养基,共进行4天的治疗。
催产素使肌上皮细胞收缩
准备新鲜的泌乳培养基,过滤,并在37°C下预热。
向泌乳培养基中添加40μg / ml重组催产素。
从吸孔中的介质不接触的Matrigel ®圆顶,并用800替换微升补充有催产素新鲜泌乳平台。
使用活细胞成像,每秒记录收缩图像120秒钟。
通过荷尔蒙戒断模仿内卷
将BOM预先加热到37°C。
从吸孔中的介质不接触的Matrigel ®圆顶,并用800替换微升新鲜BOM。
每两天用BOM更新所有培养基,共处理8天。
重装
              注意:使用预先涂有2.5%BSA的吸头。


Aspir吃的上清液和洗各孔用800两次微升冷PBS。
添加1 ml的冷PBS中,并破坏基质胶® d OME使用向上和向下运动有P 1000移液管。
检查为成功瓦解的基质胶®在显微镜下。
传送该悬浮液以15 -毫升管中并添加冷PBS至总体积10毫升。
以400 × g离心3分钟。
小心取出的上清液,重悬的器官样沉淀在新鲜的基质胶®如B1所述和板。
C.类器官处理以作进一步分析     

注意:我们建议您仔细以下之前,在显微镜下组织体的发展更新的媒体。形态与FGF2应当诱导后3支化-处理4天后,同时与BOM培养类器官只,作为所述阴性对照,应保持圆形。生乳分化Ç的确认通过CSN2 mRNA的分析使用前和治疗催乳素(D6与D10)后的qPCR,比较类器官。第i nvolution处理c的也可以确认使用qPCR的通过检测以下催乳素戒断(D10 CSN2 mRNA的表达减少与D18) ,或者在形态学水平通过分支的逐渐消失(参见图2B和图3B)。




Figu重新2.建模泌乳和复古样在原代乳腺类器官的过程。一。方案d总结了实验设计。B.泌乳和类似内卷过程中主要乳类类器官的形态。类器官形态的亮场图像以下形态发生和生乳的刺激和天4或8激素停药后。比例尺= 100μm。




图3. Passag Ë复旧状的类器官。A.描述实验设计的方案。PRL:催乳素;BOM:基础类器官介质。B.在泌乳和内卷样过程中传代的类器官的形态。明通道1(上图)和通道2(下图)的图像类器官以下形态发生和生乳的刺激和对天4或8激素停药后。比例尺= 100μm 。


RNA分离
注:嵌入在基质胶®不提取RNA的质量干扰。


吸出培养基。
甲DD 350微升RLT缓冲液(RNeasy试剂Micro试剂盒)contiaining 3.5微升β-巯基乙醇至各孔中。
用p崩解在裂解缓冲液的器官样培养erform荷兰国际集团10向上和向下运动有P 1000移液管。
Transfe r是溶液至新鲜的1.5 -毫升管中并涡旋良好。
注意:样品可以在– 80°C下保存,直到提取RNA。要进行RNA提取,请在冰上解冻样品,然后按照以下说明进行操作。


均化RNA裂解物通过执行10向上和向下以运动的单个-使用为30G胰岛素注射器。
从S tep C1 b开始,按照RNeasy Micro Kit手册中所述处理样品。
测量的使用RNA浓度纳米d ROP ™ 。
2.蛋白质提取     

注:嵌入在基质胶®干扰小号与西方印迹婷分析。请按照以下步骤删除的基质胶®之前蛋白提取。


吸培养基和离解与800 3D培养微升冷PBS补充有磷酸酶抑制剂混合物II。
转移悬浮液到一个干净1.5 -毫升在400管和离心3分钟×克,4℃。
用补充有磷酸酶抑制剂鸡尾酒II的PBS冲洗两次。
丢弃上清液,然后将沉淀重悬于100μl冰冷的即用型RIPA缓冲液中,该缓冲液中添加了蛋白酶抑制剂鸡尾酒I和磷酸酶抑制剂鸡尾酒II。
注意:小号amples可以储存在- 80℃直至蛋白质提取。要进行蛋白质提取,请在冰上解冻样品,然后按照以下说明进行操作。


超声处理的样品小号两次用60在4℃ -千赫超声波频率程序(30秒开/ 30秒关)。
涡旋样品,在冰上冷却,并重复在根据超声处理小号TEP C2即
在> 10,000 × g ,4°C下离心20分钟。
转移的上清液到一个干净的1.5 -毫升管中。
测量的使用的蛋白质浓度考马斯P rotein甲SSAY ķ它。
3.组织学的固定和嵌入     

吸出培养基和冲洗培养用800两次微升冷PBS 。
与800μl4 %PFA孵育30分钟。以下祛瘀的人的4%PFA,用PBS洗两次。
笔记:


解决方案应完全覆盖圆顶。如果需要,可以增加更大的音量。
固定的培养物可在4°C下保存在PBS中直至包埋。
制备3%的低胶凝温度琼脂糖在PBS中并在微波中慢慢融化为1.5 - 2分钟一吨1000 W(均质化每30秒通过手旋转)。
用刮刀的平坦侧面分离固定培养物,转移到含有融化的琼脂糖的塑料组织学模具中。O含更多琼脂糖。
琼脂糖固化后,unmold块。使用手术刀去除的前塞斯琼脂糖周围的基质胶® d OME在塑料包埋盒的组织学和地点。
前进到样品脱水:我ncubate在连续的1个的包埋盒- 70%H浴的EtOH ,95%EtOH中,100%的EtOH (两次),二甲苯(两次),50%二甲苯50%熔化的石蜡,和100%熔化的石蜡。
在100%熔融石蜡的第二个浴中于65°C孵育过夜。
嵌入在一个使用一个嵌入工作站组织学组织模具。
Unmold的凝固的24小时后的石蜡块。
切5 -微米的部分,并在显微镜玻片上蔓延。保持在室温下滑动,直到进一步的分析。
除去所述石蜡之前通过连续5任何染色-二甲苯的分浴池(两次),100%EtOH中(两次),95%的EtOH ,70%EtOH中,和H 2 O.


菜谱


解离溶液
注:T他的解决方案是在层流罩内准备在无菌条件和并不需要是过滤-消毒。


2 mg / ml胶原酶


2 mg / ml胰蛋白酶


5微克/毫升胰岛素


50微克/毫升庆大霉素


5%FBS


2 mM谷氨酰胺


在DMEM / F12中


BSA解决方案
注:T他的溶液可以是过滤器-消毒和当储存在4℃下重复使用多次。


PBS中2.5%的BSA


3.基底ø rganoid米edium(BOM)     

注:T他的解决方案是在层流罩内准备在无菌条件下,并不需要为过滤-消毒。


1 ×我nsulin -转铁蛋白-硒(ITS)


100 U / ml青霉素


100微克/毫升链霉素


2 mM谷氨酰胺


在DMEM / F12中


4.形态发生培养基     

注:T他的解决方案是在层流罩内准备在无菌条件下,并不需要为过滤-消毒。


BOM中的2.5 nM FGF2


5.泌乳培养基     

注:T他的解决方案是在层流罩内准备在无菌条件下,并不需要为过滤-消毒。


1微克/毫升的催乳素


1微克/毫升氢化可的松


在物料清单中


6. 4%PFA     

注:T他的解决方案是一种化学罩内准备并不必是过滤-消毒。


PBS中4%的对甲醛


7. RNA裂解缓冲液     

注:T他的解决方案是一种化学罩内准备并不必是过滤-消毒。


10微升β-巯基乙醇每1 ml的RLT裂解缓冲液(来自所述的RNeasy微型ķ它;这个解决方案可以被存储为长达一个月在室温下)。


8.磷酸酶抑制剂混合物II     

注:T他的解决方案是一种化学罩的长凳内或准备,也不需要将过滤-消毒。


2 mM咪唑


1 mM氟化钠


1.15毫钠钼酸


1个mM的钠原钒


4 mM酒石酸钠二水合物


在RIPA缓冲区中


9.蛋白酶抑制剂鸡尾酒I     

注:T他的解决方案是一种化学罩的长凳内或准备,也不需要将过滤-消毒。


500 μM AEBSF盐酸盐


150 nM抑肽酶


1 μM蛋白酶抑制剂E-64


0.5毫米EDTA


1 μM亮肽素半硫酸盐


在RIPA缓冲区中


致谢


在HL的实验室工作是由巴斯德资助,中心国家倒拉RECHERCHE的法新社国立德拉RECHERCHE (ANR-10的LabX-73和ANR-16 CE13-0017- 01),基金会ARC(PJA 20161205028和20181208231 ),计划Barrande ,以及AFM-马拉松式节目基金会。AC由Revive财团的博士后研究金资助。EC由一位博士资助。来自索邦大学的研究金。ZK是由马萨里克大学(MUNI / G /二千〇一十八分之一千四百四十六),移动性补助由教育部的资助局资助和青年和体育,并从医学MU的学院资金的初级研究员ROZV / 28(/ LF / 2020)。JS由P-Pool(MU大学医学院)和Masaryk University的Grant Agency(MUNI / A / 1565/2018)资助。


该方案源自原始研究论文“泌乳和复旧的原始乳腺类器官模型” (Sumbal等人,2020b)。


利益争夺


作者宣称他们没有竞争利益。


伦理


动物研究是审查和符合欧共体理事会指令(A 75-15-01-3)和规定法国法律认可的研究所巴斯德动物保护委员会(CETEA)。


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引用:Charifou, E., Sumbal, J., Koledova, Z., Li, H. and Chiche, A. (2021). A Robust Mammary Organoid System to Model Lactation and Involution-like Processes. Bio-protocol 11(8): e3996. DOI: 10.21769/BioProtoc.3996.
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