We thank Ben Abrams for technical assistance and core support from the University of California, Santa Cruz (UCSC) Institute for the Biology of Stem Cells (IBSC). We thank Susan Strome and Bill Saxton for the use of their Solamere Spinning Disk Confocal Microscope. This work was supported in part by grants to UCSC from the Howard Hughes Medical Institute through the James H. Gilliam Fellowships for Advanced Study program (S.R.), from the NIH (NIH GM058903) for the initiative for maximizing student development (H.M.) and from the National Science Foundation for a graduate research fellowship (O.C. DGE 1339067) and by a grant (A18-0370) from the UC-Cancer Research Coordinating Committee (LH).

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, Santa Cruz.
1. Day 1: Mammary gland digestion
<ol>
	<li>Prepare to harvest the MGs from mature female mice 10-14 weeks of age.
	<ol>
		<li>Perform the harvesting on an open bench under aseptic conditions.</li>
		<li>Sterilize all surgical supplies, cork boards, and pins by autoclaving and soaking in 70% alcohol for 20 min prior to surgery.</li>
		<li>Anesthetize animals with sodium pentobarbital (2X anesthetic dose of 0.06 mg/g body weight) delivered via intraperitoneal injection with a 0.5 mL insulin syringe.</li>
		<li>Monitor the level of anesthesia by pinching the animal&#39;s toes to check for a reflex response and commence the protocol only after the animal is fully anesthetized.</li>
		<li>Place the animal on its back, pin its appendages to the corkboard, and wipe down its abdomen and chest with ethanol.</li>
	</ol>
	</li>
	<li>To harvest the #2, 3, 4, and 5 MGs from one mouse (i.e., 8 MGs, Figure 1A), identify the midline between the two hind legs and make a small incision (1 cm) on the abdominal skin with sharp scissors, then extend the cut up to the neck18.</li>
	<li>Follow by making small cuts laterally towards the legs and arms to allow for the release of the skin using a cotton swab. Pull the skin away and stretch it tight before pinning it down on one side (Figure 1B, step 1)18. Remove the MGs by cutting under them, and remove the lymph nodes from the #4 glands (Figure 1B, steps 2, 3)18. Repeat the procedure on the other side of the body.</li>
	<li>Collect the MG tissue in 50 mL of 4 &#176;C Dulbecco&#39;s Modified Eagle&#39;s Media (DMEM)/Nutrient Mixture F12 (F12) supplemented with 5% fetal bovine serum (FBS) and 1X Antibiotic-Antimycotic (Anti-Anti)18.</li>
	<li>Chop the glands in a 35 mm dish or on a ceramic plate using a razor blade or tissue chopper. Rotate the plate every five manual chops or every round on the tissue chopper until the tissue pieces can fit through a 1 mL micropipette tip with ease (~0.1 mm/fragment, Figure 1C).</li>
	<li>Digest the MGs in Digestion Medium (see Table 1) for 14 h in a 6 well low adhesion dish at 37 &#176;C, 5% CO2.</li>
</ol>
2. Day 2: Isolation of mammary epithelial tissue fragments
<ol>
	<li>After 14 h of digestion, gently mix by pipetting the digested tissue 10X using a 1 mL micropipette to break down any remaining stroma or adipose tissue, ensuring that neither bubbles nor excess mechanical force are generated. 
	NOTE: If there is incomplete digestion after 14 h, this could be due to the accumulation of sheared DNA. In this case, add 1 &#181;L of 1 mg/mL deoxyribonuclease I (DNase I) per 2 mL of Digestion Medium. Incubate for another 30 min at 37 &#176;C, 5% CO2.</li>
	<li>Collect tissue in a 15 mL tube and rinse the well used for digestion with 2-3 mL of tissue culture grade 1X Dulbecco&#39;s Phosphate Buffered Saline (DPBS) free of Ca2+ and Mg2+. Centrifuge at 600 x g for 10 min.</li>
	<li>Evacuate the supernatant containing the lipid layer and medium, and then resuspend the pellet in 5 mL of DPBS and switch to a new 15 mL tube. Centrifuge at 600 x g for 10 min.</li>
	<li>During centrifugation place a 70 &#181;m nylon cell strainer in a 50 mL tube and prewet the strainer using 10 mL of 37 &#176;C DMEM/F12.</li>
	<li>Resuspend tissue fragments from protocol step 2.4 using 5 mL of DPBS and pass the suspension through a prewet 70 &#181;m nylon cell strainer to remove stromal cells and single cells (Figure 1D).</li>
	<li>Collect the tissue fragments on the cell strainer. Rinse 4X with 10 mL of 37 &#176;C DMEM/F12 (Figure 1D). 
	CAUTION: Incomplete rinsing will result in cultures contaminated with non-epithelial cells.</li>
	<li>Release the tissue fragments by holding the strainer tab with gloved fingers, inverting the strainer over a 60 mm tissue culture dish and passing 1 mL aliquots of Maintenance Medium (see Table 1) through the bottom of the strainer 4X (Figure 1E).</li>
	<li>Check the strainer for tissue fragment remnants, which will be visible by the naked eye, and rinse the strainer 1X more with 1 mL of Maintenance Medium if any fragments are still adhering to the strainer. The rinsed tissue fragments should now be free of stromal cells.</li>
	<li>Quickly examine the 60 mm dish containing the MG fragments from protocol step 2.9 under an inverted microscope (4X or 10X objective, Figure 1F). A typical preparation of eight MGs yields ~500 fragments. Look for single cells or fat droplets and whether there are contaminating cells. 
	NOTE: If there are contaminating cells, repeat the filtration step by collecting the medium and fragments from the 60 mm dish using a 5 mL pipette and filtering the fragment again through a fresh 70 &#181;m strainer, repeating protocol steps 2.6, 2.8-2.10.</li>
	<li>Incubate 24 h at 37 &#176;C, 5% CO2, allowing the tissue fragment to adhere and generate bilayered fragments (Figure 2A). 
	NOTE:If the fragments have not settled by 24 h, continue to incubate until adhered. If the fragments do not adhere well, the separation of the cell compartments will not work. If researchers are concerned about adhesion, the tissue culture plates can be treated to promote fragment attachment (e.g., poly-L-lysine).</li>
</ol>
3. Day 3: Differential trypsinization of myoepithelial and luminal epithelial cells
<ol>
	<li>To separate MyoECs from LECs, empty the media from the dish, rinse 1x with 1 mL of DPBS and add 1 mL of fresh trypsin-EDTA (0.05%), and carefully monitor the digestion under an inverted microscope (10X or 20X objective, Figure 2B,C,F). The detachment of the outer MyoEC layer will require 3-6 min, depending on trypsin-EDTA (0.05%) strength. 
	NOTE: Please see Figure 2 for representative images showing this process. Under brightfield illumination, the MyoECs appear rounded and have a brighter appearance in comparison to the LECs, which remain adhered in the center and appear darker.</li>
	<li>Collect the MyoEC fraction in a 15 mL tube containing 2 mL 10% FBS/DPBS. Without disturbing the LECs, gently rinse the 60 mm dish with 2 mL of DPBS and then dispose of the DPBS (Figure 2H-I). 
	NOTE: The usual recovery for MyoECs is within a range of (3.5 x 106-1.5 x 106) depending on the size of the MGs.</li>
	<li>To remove the LEC fraction, add 1 mL of trypsin-EDTA (0.05%) to the dish again and incubate 7-15 min, monitoring carefully to prevent overdigestion. Quench the trypsin-EDTA (0.05%) on the dish with 2 mL of 10% FBS/DPBS. Collect the LEC fraction in a new 15 mL tube. 
	NOTE: The usual recovery for LECs is within a range (2 x 106-4.2 x 106) depending on the size of the MGs. Routinely, the purity of both fractions as assayed by immunohistochemistry is ~90%19 (Figure 2E).</li>
	<li>Centrifuge each fraction for 5 min at 300 x g to remove the trypsin-EDTA (0.05%). Resuspend the pellet in 250 &#181;L of Maintenance Medium and count each cell population using a hemocytometer or automated cell counter. Place the cells on ice while counting. 
	NOTE: If genetic manipulation of the cell fractions is desired, primary cells can be grown on low adhesion dishes and infected with lentivirus20.</li>
</ol>
4. Day 3: Combining and embedding cell fractions in an extracellular matrix
NOTE: Once the MyoEC and LEC fractions have been collected and counted, they can be combined. The typical MyoEC/LEC ratio is 1:3 (Figure 3A)19. Different studies can be performed. For example, to perform mosaic studies, fractions can be generated from wild type (WT) and mutant (Mut) mice and combined (MyoEC/LEC: WT/WT; WT/Mut; Mut/WT; Mut/Mut)21, or fractions can be combined using different ratios of MyoECs/LECs19.
<ol>
	<li>Based on cell counts, calculate the number of wells (8 well chamber, see Table of Materials) that need to be prepared for 12,000 cells/well (e.g. 3,000 MyoECs:9,000 LECs).</li>
	<li>Establish the base layer for 3D culture by adding 90 &#181;L of 50% ECM (50% ECM/50% DMEM/F12, without phenol red - see the Table of Materials) to each well. Ensure there are no bubbles and the wells are coated evenly. To solidify the base layer, incubate the slides at 37 &#176;C, 5% CO2 for 30 min. 
	NOTE: The ECM needs to stay ice-cold until this step, otherwise it will polymerize prematurely and lead to uneven base coating and polymerization. Using a base ECM enhances organoid growth in a single plane, which aids image capture. This also obtains faster organoid growth and uses less ECM22.</li>
	<li>During polymerization, prepare the cell mixes. Pellet the MyoEC and LEC fractions at 300 x g for 5 min and resuspend each cell fraction in 10% ECM/90% Growth Medium so each well has 100 &#181;L (see Table 1 for how to make Growth Medium). 
	NOTE: For ease of preparation, replicate wells using the same cell mixes. These can be combined and prepared in one tube (e.g., four wells of the same cell mix can be prepared in 400 &#181;L of 10% ECM/90% Growth Medium).</li>
	<li>Add 100 &#181;L of each cell mix in 10% ECM/90% Growth Medium to each well and allow organoids to settle for 20 min at 37 &#176;C, 5% CO2.</li>
	<li>Once the cells have settled, gently add 100 &#181;L of Growth Medium by gently pipetting down the chamber wall of each well. Incubate the slides at 37 &#176;C, 5% CO2 (see Figure 3A for the total composition of each well). 
	NOTE: The Rho Kinase inhibitor, R-spondin, and Nrg1 are factors that have been identified as important for the long-term culture of organoids grown from both primary murine mammary cells and human breast cancer cells13,14. In addition, the stem cell factors B27 and N2 extend the time that organoids can be cultured.</li>
	<li>Image cells every 24 h to track their growth and gently renew the Growth Medium every 2-3 days using 100 &#181;L/well (Figure 3B-E). 
	NOTE: Use extreme care when renewing the medium. Tilt the chamber slides to collect the medium at one corner of the wells. Remove &#8804;100 &#181;L of the medium and replenish with care to leave the ECM layer undisturbed.</li>
	<li>If researchers are interested in investigating lactation/alveologenesis, switch to Alveologenesis Medium (see Table 1) on day 5 and continue to renew the medium every 2-3 days until day 10 (Figure 3F) or beyond by passaging using recovery solution (see the Table of Materials)13. 
	NOTE: At this point, organoids can be isolated from the ECM by incubating at 4 &#176;C in 400 &#181;L of 4 &#176;C recovery solution (see Table of Materials for protocol and reagent info).</li>
</ol>
5. Day 5 or 10: Fixing and immunostaining organoids
<ol>
	<li>Remove the medium carefully by gently pipetting off the media (a bulb pipette works best). Rinse each well using 200 &#181;L of 1x Dulbecco&#39;s Phosphate Buffered Saline (DPBS, see recipes).</li>
	<li>Fix the organoids using cold (4 &#176;C) 4% (w/v) paraformaldehyde (PFA, see recipes) for 10 min at room temperature. 
	CAUTION: PFA is hazardous. Wear personal protective equipment (lab coat, gloves, and safety glasses). This step should be performed inside a fume hood. 
	NOTE: The ECM is dissolved by the PFA treatment. Incomplete ECM removal can lead to background staining when the organoids are analyzed by immunofluorescence.</li>
	<li>Remove the 4% PFA and add 200 &#181;L of 0.2% (w/v) glycine/DPBS (see Table 1) to each well. Incubate the slides at room temperature for 30 min or 4 &#176;C overnight on a rocking surface set to a slow setting. 
	NOTE: The organoids can be stored for 1-3 days in DPBS at 4 &#176;C prior to the next step.</li>
	<li>Permeabilize the organoids using DPBS + 0.25% Triton X-100 (PBST, see Table 1) for 10 min at room temperature.</li>
	<li>Block the organoids using 5% donkey serum (DS) (or other serum matching the species of the secondary antibody) in DPBS for 1 h on a rocking surface. 
	NOTE: This step can be performed overnight at 4 &#176;C on a rocking surface.</li>
	<li>Prepare primary antibodies in 1% DS/DPBS. Use 125-200 &#181;L for each well. Perform immunostaining by incubating the organoids in primary antibody overnight at 4 &#176;C on a rocking surface.</li>
</ol>
6. Day 11: Complete immunofluorescence
<ol>
	<li>Wash each well 2X with 200 &#181;L PBST for 5 min. Add secondary antibody in 1% DS/DPBS using 125-200 &#181;L for each well. Incubate at room temperature on a rocking surface for 45 min.</li>
	<li>Wash each well 2X using 200 &#181;L PBST per well.</li>
	<li>Stain the nuclei using Hoechst DNA dye in DPBS (1:2,000 in DPBS) for 5 min at room temperature.</li>
	<li>Remove all liquid left on the well by gently suctioning with a vacuum.</li>
	<li>Carefully remove the chambers and gasket, place one drop (~30 &#181;L) of mounting media (see Table of Materials) on each well and coverslip, taking care to remove bubbles. Allow the slide to dry in a dark space for 1-2 days. Seal with clear nail polish. Image the organoids on a confocal microscope (Figure 3E-F).</li>
</ol>

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The authors have nothing to disclose.

Here, a method is presented detailing how researchers can generate 3D organoid cultures using primary MG cells. The difference between this and other protocols is that we detail a method to separate the two, distinct MG cell compartments: the outer basal MyoECs and inner LECs. Our method employs a two-step trypsin-EDTA (0.05%) treatment that we call differential trypsinization19. This procedure allows researchers to isolate MyoECs and LECs without using sophisticated flow cytometry and thus can be...

The mammary gland (MG) is a tree-like, tubular epithelial structure embedded within an adipocyte rich stroma. The bilayered ductal epithelium comprises an outer, basal layer of contractile, myoepithelial cells (MyoECs) and an inner layer of luminal, secretory epithelial cells (LECs), encircling a central lumen1. During lactation when the outer MyoECs contract to squeeze milk from the inner alveolar LECs, the MG undergoes numerous changes that are under the control of growth factors (e.g., EGF and FGF) and hormones (e.g. progesterone, insulin, and prolactin). These changes cause the differentiation of specialized structures, alveoli, which synthesize and secrete milk during lactation1. The mammary epithelia can be experimentally manipulated using techniques in which either epithelial tissue fragments, cells, or even a single basal cell are transplanted into host mammary fat pads, precleared of endogenous mammary parenchyma, and allowed to grow out to reconstitute an entire, functional epithelial tree2,3,4,5. Transplantation is a powerful technique, but it is time-consuming and impossible if a mutation results in early embryonic lethality (prior to E14) that prevents the rescue of transplantable mammary anlage. Furthermore, investigators frequently wish to research the roles of the two different compartments, which are derived from lineage-restricted progenitor cells. While Cre-lox technology allows differential genetic manipulation of MyoECs and LECs, this is also a time-consuming and expensive undertaking. Thus, since the 1950s, investigators have used in vitro mammary organoids as a relatively easy and efficient way to address questions concerning mammary tissue structure and function6,7.
In early protocols describing the isolation and culture of primary mammary epithelial cells, investigators found that a basement membrane matrix (BME), composed of a plasma clot and chicken embryo extract, was required for MG fragments grown on a dish6. In the following decades, extracellular matrices (ECMs, collagen, and jellylike protein matrix secreted by Engelbreth-Holm-Swarm murine sarcoma cells) were developed to facilitate 3D culture and better mimic the in vivo environment7,8,9,10. Culturing cells in 3D matrices revealed by multiple criteria (morphology, gene expression, and hormone responsiveness) that such a microenvironment better models in vivo physiological processes9,10,11,12. Research using primary murine cells identified key growth factors and morphogens necessary for the extended maintenance and differentiation of organoids13. These studies have set the stage for the protocol presented here, and for the culture of human breast cells as 3D organoids, which is now a modern clinical tool, allowing for drug discovery and drug testing on patient samples14. Overall, organoid culturing highlights the self-organization capacities of primary cells and their contributions to morphogenesis and differentiation.
Presented here is a protocol to culture murine epithelia that can be differentiated into milk-producing acini. A differential trypsinization technique is used to isolate the MyoECs and LECs that comprise the two distinct MG cell compartments. These separated cell fractions can then be genetically manipulated to overexpress or knockdown gene function. Because lineage-intrinsic, self-organization is an innate property of mammary epithelial cells15,16,17, recombining these cell fractions allows researchers to generate bilayered, mosaic organoids. We begin by enzymatically digesting the adipose tissue, and then incubating the mammary fragments on a tissue culture dish for 24 h (Figure 1). The tissue fragments settle on polystyrene dishes as bilayered fragments with their in vivo organization: outer myoepithelial layer surrounding inner luminal layers. This cellular organization allows for the isolation of the outer MyoECs by trypsin-EDTA (0.05%) treatment for 3-6 min followed by a second round of trypsin-EDTA (0.05%) treatment that detaches the remaining inner LECs (Figure 2). Thus, these cell types with different trypsin sensitivity are isolated and can subsequently be mixed and plated in ECM (Figure 3). The cells undergo self-organization to form bilayered spheres, comprising an outer layer of MyoECs surrounding inner LECs. Lumen formation occurs as the cells grow in a medium containing a cocktail of growth factors (see recipes for Growth Medium)13. After 5 days, organoids can be differentiated into milk-producing acini by switching to Alveologenesis Medium (see recipes and Figure 3F) and incubated for another 5 days. Alternatively, organoids will continue to expand and branch in Growth Medium for at least 10 days. Organoids can be analyzed using immunofluorescence (Figure 3D-F) or released from the ECM using a recovery solution (see Table of Materials) and analyzed via other methods (e.g., immunoblot, RT-qPCR).

<table><tbody><tr><td>15 ml High-Clarity Polypropylene Conical Tube (BD Falcon)</td><td>Fisher Scientific</td><td>352096</td><td></td></tr><tr><td>24 well ultra-low attachment plate (Corning)</td><td>Fisher Scientific</td><td>CLS3473-24EA</td><td></td></tr><tr><td>35 mm TC-treated Easy-Grip Style Cell Culture Dish (BD Falcon)</td><td>Fisher Scientific</td><td>353001</td><td></td></tr><tr><td>50 ml High-Clarity polypropylene conical tube (BD Falcon)</td><td>Fisher Scientific</td><td>352098</td><td></td></tr><tr><td>60 mm TC-treated Easy-Grip Style Cell Culture Dish (BD Falcon)</td><td>Fisher Scientific</td><td>353004</td><td></td></tr><tr><td>70 &amp;micro;M nylon cell strainer (Corning)</td><td>Fisher Scientific</td><td>08-771-2</td><td></td></tr><tr><td>Antibiotic-Antimycotic (100X)</td><td>Thermo Fisher Scientific</td><td>15240062</td><td>Pen/Strep also works</td></tr><tr><td>B27 supplement without vitamin A (50x)</td><td>Thermo Fisher Scientific</td><td>12587010</td><td></td></tr><tr><td>B6 ACTb-EGFP mice</td><td>The Jackson Laboratory</td><td>003291</td><td></td></tr><tr><td>BD Insulin syringe 0.5 mL</td><td>Thermo Fisher Scientific</td><td>14-826-79</td><td></td></tr><tr><td>Class 2 Dispase (Roche)</td><td>Millipore Sigma</td><td>4942078001</td><td></td></tr><tr><td>Class 3 Collagenase</td><td>Worthington Biochemical</td><td>LS004206</td><td></td></tr><tr><td>Corning Cell Recovery solution</td><td>Fisher Scientific</td><td>354253</td><td>Follow the guidelines for use &amp;ndash; Extraction of Three-Dimensional Structures&lt;br/&gt; from Corning Matrigel Matrix</td></tr><tr><td>Corning Costar Ultra-Low Attachment 6-well</td><td>Fisher Scientific</td><td>CLS3471</td><td></td></tr><tr><td>Dexamethasone</td><td>Millipore Sigma</td><td>D4902-25MG</td><td></td></tr><tr><td>DMEM/F12, no phenol red</td><td>Thermo Fisher Scientific</td><td>11039-021</td><td></td></tr><tr><td>DNase (Deoxyribonuclease I)</td><td>Worthington Biochemical</td><td>LS002007</td><td></td></tr><tr><td>Donkey anti-Goat 647</td><td>Thermo Fisher Scientific</td><td>A21447</td><td>Use at 1:500, Lot: 1608641, stock 2 mg/mL, RRID:AB_2535864</td></tr><tr><td>Donkey anti-Mouse 647</td><td>Jackson ImmunoResearch</td><td>715-606-150</td><td>Use at 1:1000, Lot: 140554, stock 1.4 mg/mL</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12)</td><td>Thermo Fisher Scientific</td><td>11330-057</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline (DPBS)</td><td>Thermo Fisher Scientific</td><td>14190-250</td><td>Without Mg2+/Ca2+</td></tr><tr><td>EGF</td><td>Fisher Scientific</td><td>AF-100-15-100ug</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>VWR</td><td>97068&amp;ndash;085</td><td>100% US Origin, premium grade, Lot: 059B18</td></tr><tr><td>Fluoromount-G (Southern Biotech)</td><td>Fisher Scientific</td><td>0100-01</td><td>Referred to as mounting media in text</td></tr><tr><td>Gentamicin</td><td>Thermo Fisher Scientific</td><td>15710064</td><td></td></tr><tr><td>Glycine</td><td>Fisher Scientific</td><td>BP381-5</td><td></td></tr><tr><td>Goat anti-WAP</td><td>Santa Cruz Biotech</td><td>SC-14832</td><td>Use at 1:250, Lot: J1011, stock 200 &amp;micro;g/mL, RRID:AB_677601</td></tr><tr><td>Hoechst 33342</td><td>AnaSpec</td><td>AS-83218</td><td>Use 1:2000, stock is 20mM</td></tr><tr><td>Insulin</td><td>Millipore Sigma</td><td>I6634-100mg</td><td></td></tr><tr><td>KCl</td><td>Fisher Scientific</td><td>P217-500</td><td></td></tr><tr><td>KH2PO4</td><td>Fisher Scientific</td><td>P285-500</td><td></td></tr><tr><td>KRT14&amp;ndash;CreERtam</td><td>The Jackson Laboratory</td><td>5107</td><td></td></tr><tr><td>Matrigel Growth Factor Reduced (GFR); Phenol Red-Free; 10 mL</td><td>Fisher Scientific</td><td>CB-40230C</td><td>Lot: 8204010, stock concentration 8.9 mg/mL</td></tr><tr><td>MillexGV Filter Unit 0.22 &amp;micro;m</td><td>Millipore Sigma</td><td>SLGV033RS</td><td></td></tr><tr><td>Millicell EZ SLIDE 8-well glass, sterile</td><td>Millipore Sigma</td><td>PEZGS0816</td><td>These chamber slides are great for gasket removal but other brands can work well (e.g. Lab Tek II).</td></tr><tr><td>Mouse anti-SMA</td><td>Millipore Sigma</td><td>A2547</td><td>Use at 1:500, Lot: 128M4881V, stock 5.2 mg/mL, RRID:AB_476701</td></tr><tr><td>N-2 Supplement (100x)</td><td>Thermo Fisher Scientific</td><td>17502048</td><td></td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>S671-3</td><td></td></tr><tr><td>NaH2PO4</td><td>Fisher Scientific</td><td>S468-500</td><td></td></tr><tr><td>Nrg1</td><td>R&amp;amp;D</td><td>5898-NR-050</td><td></td></tr><tr><td>Ovine Pituitary Prolactin</td><td>National Hormone and Peptide Program</td><td></td><td>Purchased from Dr. Parlow at Harbor-UCLA Research and Education Institute</td></tr><tr><td>Paraformaldahyde</td><td>Millipore Sigma</td><td>PX0055-3</td><td></td></tr><tr><td>Pentobarbital</td><td>Millipore Sigma</td><td>P3761</td><td></td></tr><tr><td>R26R-EYFP</td><td>The Jackson Laboratory</td><td>6148</td><td></td></tr><tr><td>Rho inhibitor Y-27632</td><td>Tocris</td><td>1254</td><td></td></tr><tr><td>R-spondin</td><td>Peprotech</td><td>120-38</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Fisher Scientific</td><td>S318-500</td><td></td></tr><tr><td>Sterile Filtered Donkey Serum</td><td>Equitech-Bio Inc.</td><td>SD30-0500</td><td></td></tr><tr><td>Sterile Filtered Donkey Serum</td><td>Equitech-Bio Inc.</td><td>SD30-0500</td><td></td></tr><tr><td>Triton X-100</td><td>Millipore Sigma</td><td>x100-500ML</td><td>Laboratory grade</td></tr><tr><td>Trypsin EDTA 0.05%</td><td>Thermo Fisher Scientific</td><td>25300-062</td><td></td></tr></tbody></table>

generation of mosaic mammary organoids by differential trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The protocol presented here describes a method for investigating specific lineage contributions of mammary epithelial cells by making use of mosaic organoids. To obtain primary murine cells for organoids, the mammary gland epithelium must first be isolated from the surrounding adipocyte rich stroma (Figure 1). This process is described briefly here and is also described in a previously published study18. To obtain enough cells, it is r...

Watch this Scientific Journal Video about Generation of Mosaic Mammary Organoids by Differential Trypsinization at JoVE.com

Generation of Mosaic Mammary Organoids by Differential Trypsinization

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

generation-of-mosaic-mammary-organoids-by-differential-trypsinization

Research

JoVE Journal

Developmental Biology

6.2K Views.  University of California, Santa Cruz. The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Video: Generation of Mosaic Mammary Organoids by Differential Trypsinization

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal mammary epithelial cell (NAMEC)-derived extracellular vesicles can be isolated from NAMEC medium via differential ultracentrifugation. Based on their density, EVs can be purified via ultracentrifugation at 110,000 x g. The EV preparation from ultracentrifugation can be further separated using a continuous density gradient to prevent contamination with soluble proteins. The purified EVs can then be further evaluated using nanoparticle-tracking analysis, which measures the size and number of vesicles in the preparation. The extracellular vesicles with a size ranging from 50 to 150 nm are exosomes. The NAMEC-derived EVs/exosomes can be ingested by mammary epithelial cells, which can be measured by flow cytometry and confocal microscopy. Some mammary stem cell properties (e.g., mammary gland-forming ability) can be transferred from the stem-like NAMECs to mammary epithelial cells via the NAMEC-derived EVs/exosomes. Isolated primary EpCAMhi/CD49flo luminal mammary epithelial cells cannot form mammary glands after being transplanted into mouse fat pads, while EpCAMlo/CD49fhi basal mammary epithelial cells form mammary glands after transplantation. Uptake of NAMEC-derived EVs/exosomes by EpCAMhi/CD49flo luminal mammary epithelial cells allows them to generate mammary glands after being transplanted into fat pads. The EVs/exosomes derived from stem-like mammary epithelial cells transfer mammary gland-forming ability to EpCAMhi/CD49flo luminal mammary epithelial cells.

This protocol describes methods for purifying, quantitating, and characterizing extracellular vesicles (EVs)/exosomes from non-adherent/mesenchymal mammary epithelial cells and for using them to transfer mammary gland-forming ability to luminal mammary epithelial cells. EVs/exosomes derived from stem-like mammary epithelial cells can transfer this cell property to cells that ingest the EVs/exosomes.

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal ...

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

Medicine

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids represent useful models to study the mechanisms that control stem cell self-renewal and differentiation in mammals, including primary ciliogenesis and ciliary signaling. Primary ciliogenesis is the dynamic process of assembling the primary cilium, a key cell signaling center that controls stem cell self-renewal and/or differentiation in various tissues. Here we present a comprehensive protocol for the immunofluorescence staining of cell lineage and primary cilia markers, in whole-mount mouse mammary organoids, for light sheet microscopy. We describe the microscopy imaging method and an image processing technique for the quantitative analysis of primary cilium assembly and length in organoids. This protocol enables a precise analysis of primary cilia in complex three-dimensional structures at the single cell level. This method is applicable for immunofluorescence staining and imaging of primary cilia and ciliary signaling in mammary organoids derived from normal and genetically modified stem cells, from healthy and pathological tissues, to study the biology of the primary cilium in health and disease.

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

Stem cell-derived organoids facilitate the analysis of molecular and cellular processes that regulate stem cell self-renewal and differentiation during organogenesis in mammalian tissues. Here we present a protocol for the analysis of the biology of the primary cilium in mouse mammary organoids.

Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids ...

Genetics

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.

This protocol discusses an approach for generating epithelial organoids from primary normal and tumor mammary tissue through differential centrifugation. Furthermore, instructions are included for three-dimensional culturing as well as immunofluorescent imaging of embedded organoids.

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after ...

Cancer Research

Establishment and Culture of Patient-Derived Breast Organoids

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor organoids developed in our laboratory consist of a mix of multiple tumor-derived cell populations, and thus represent a better approximation of tumor cell diversity and milieu than the established 2D cancer cell lines. Organoids serve as an ideal in vitro model, allowing for cell-extracellular matrix interactions, known to play an important role in cell-cell interactions and cancer progression. Patient-derived organoids also have advantages over mouse models as they are of human origin. Furthermore, they have been shown to recapitulate the genomic, transcriptomic as well as metabolic heterogeneity of patient tumors; thus, they are capable of representing tumor complexity as well as patient diversity. As a result, they are poised to provide more accurate insights into target discovery and validation and drug sensitivity assays. In this protocol, we provide a detailed demonstration of how patient-derived breast organoids are established from resected breast tumors (cancer organoids) or reductive mammoplasty-derived breast tissue (normal organoids). This is followed by a comprehensive account of 3D organoid culture, expansion, passaging, freezing, as well as thawing of patient-derived breast organoid cultures.

A detailed protocol is provided here for establishing human breast organoids from patient-derived breast tumor resections or normal breast tissue. The protocol provides comprehensive step-by-step instructions for culturing, freezing, and thawing human patient-derived breast organoids.

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor ...

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed in order to create testicular organoids. Many of these methods rely upon extracellular matrix (ECM) to promote de novo tissue assembly, however, there are differences between methods in terms of biomimetic morphology and function of tissues. Moreover, there are few direct comparisons of published methods. Here, a direct comparison is made by studying differences in organoid generation protocols, with provided outcomes. Four archetypal generation methods: (1) 2D ECM-free, (2) 2D ECM, (3) 3D ECM-free, and (4) 3D ECM culture are described. Three primary benchmarks were used to assess the testicular organoid generation. These are cellular self-assembly, inclusion of major cell types (Sertoli, Leydig, germ, and peritubular cells), and appropriately compartmentalized tissue architecture. Of the four environments tested, 2D ECM and 3D ECM-free cultures generated organoids with internal morphologies most similar to native testes, including the de novo compartmentalization of tubular versus interstitial cell types, the development of tubule-like-structures, and an established long-term endocrine function. All methods studied utilized unsorted, primary murine testicular cell suspensions and used commonly accessible culture resources. These testicular organoid generation techniques provide a highly accessible and reproducible toolkit for research initiatives into testicular organogenesis and physiology in vitro.

Here, four methods for generating testicular organoids from primary neonatal murine testicular cells are described i.e., extracellular matrix (ECM) and ECM-free 2D and 3D culture environments. These techniques have multiple research applications and are especially useful for studying testicular development and physiology in vitro.

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed ...

Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...

Bioengineering

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The three-dimensional culture method described in this protocol recapitulates pancreas development from dispersed embryonic mouse pancreas progenitors, including their substantial expansion, differentiation and morphogenesis into a branched organ. This method is amenable to imaging, functional interference and manipulation of the niche.

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

Biology

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for &#62;6 months.

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Summary

Explore More Videos

Chapters in this video

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

Establishment and Culture of Patient-Derived Breast Organoids

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

Growth and Characterization of Irradiated Organoids from Mammary Glands

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres