Name: generation of mosaic mammary organoids by differential trypsinization
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Description: Watch this Scientific Journal Video about Generation of Mosaic Mammary Organoids by Differential Trypsinization at JoVE.com

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>Anest...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Desmosomal+adhesion+regulates+epithelial+morphogenesis+and+cell+positioning.">Desmosomal adhesion regulates epithelial morphogenesis and cell positioning.</a> Nature Cell Biology. 3 (9), 823-830 (2001).</li><li>Chanson, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-organization+is+a+dynamic+and+lineage-intrinsic+property+of+mammary+epithelial+cells.">Self-organization is a dynamic and lineage-intrinsic property of mammary epithelial cells.</a> Proceedings of the National Academy of Science U S A. 108 (8), 3264-3269 (2011).</li><li>Honvo-Houeto, E., Truchet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indirect+Immunofluorescence+on+Frozen+Sections+of+Mouse+Mammary+Gland.">Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland.</a> Journal of Visualized Experiments. (106), e53179 (2015).</li><li>Macias, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SLIT/ROBO1+signaling+suppresses+mammary+branching+morphogenesis+by+limiting+basal+cell+number.">SLIT/ROBO1 signaling suppresses mammary branching morphogenesis by limiting basal cell number.</a> Developmental Cell. 20 (6), 827-840 (2011).</li><li>Welm, B. E., Dijkgraaf, G. J., Bledau, A. S., Welm, A. L., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+transduction+of+mammary+stem+cells+for+analysis+of+gene+function+during+development+and+cancer.">Lentiviral transduction of mammary stem cells for analysis of gene function during development and cancer.</a> Cell Stem Cell. 2 (1), 90-102 (2008).</li><li>Smith, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VANGL2+regulates+luminal+epithelial+organization+and+cell+turnover+in+the+mammary+gland.">VANGL2 regulates luminal epithelial organization and cell turnover in the mammary gland.</a> Scientific Reports. 9 (1), 7079 (2019).</li><li>Lee, G. Y., Kenny, P. A., Lee, E. H., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+culture+models+of+normal+and+malignant+breast+epithelial+cells.">Three-dimensional culture models of normal and malignant breast epithelial cells.</a> Nature Methods. 4 (4), 359-365 (2007).</li><li>Campbell, J. J., Davidenko, N., Caffarel, M. M., Cameron, R. E., Watson, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multifunctional+3D+co-culture+system+for+studies+of+mammary+tissue+morphogenesis+and+stem+cell+biology.">A multifunctional 3D co-culture system for studies of mammary tissue morphogenesis and stem cell biology.</a> PLoS One. 6 (9), e25661 (2011).</li><li>Labarge, M. A., Garbe, J. C., Stampfer, M. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#181;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 &#8211; Extraction of Three-Dimensional Structures 
			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&#39;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&#39;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&#8211;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 &#181;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&#8211;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 &#181;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&#38;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 ...

Our method is significant because it allows researchers to isolate and manipulate different cell types for investigation of their individual lineage contributions to tissue form and function. This technique is a gentle and efficient method for separating cell types and as a result, the integrity of the cells is preserved for 3D culture. This technique can also be applied to other systems to isolate cells that don't have well-characterized biomarkers.To harvest the number two, three, four, and five mammary glands from a 10 to 14-week-old female mouse, first make a one centimeter incision at the midline between the two hindlimbs. Extend the cut up to the neck and make small lateral cuts from the midline incision toward the limbs. Then stretch the skin taught before securing it with a pin on each side of the animal.Using forceps, collect the lymph nodes from the number four glands. To remove the mammary glands, use sharp scissors to cut under each region of tissue, placing the tissues in 50 milliliters of four degrees Celsius DMEM-F12 supplemented with 5%FBS and antibiotic-antimycotic as they are harvested. When all of the tissues have been collected, chop the glands until the pieces can fit easily through a one milliliter pipette tip rotating the plate as necessary so that all of the tissues are minced evenly.Then transfer the fragments in three milliliters of digestion medium into a single well of a six-well low adhesion dish for 14 hours at 37 degrees Celsius and 5%carbon dioxide. The next morning, gently pipette the tissues 10 times with a one milliliter micropipette and transfer the tissue fragments into a 15 milliliter tube. Collect the tissue by centrifugation and resuspend the pellet in five milliliters of fresh DPBS.Filter the suspension through a pre-wet 70 micrometer nylon cell strainer, rinsing the tube in the strainer four times with 10 milliliters of 37 degrees Celsius DMEM-F12 per wash. To release the tissue fragments, hold the strainer tab with gloved fingers and invert the strainer over a 60 millimeter tissue culture dish. Pass four one milliliter aliquots of maintenance medium through the bottom of the strainer and quickly examine the dish for single cells, fat droplets, and contaminating tissue under an inverted microscope.Then place the plate in the cell culture incubator for 24 hours to allow the tissue fragments to adhere to the dish and to generate bilayered fragments. To separate the myoepithelial cells from the luminal epithelial cells, rinse the dish with one milliliter of DPBS before adding one milliliter of fresh 0.5%trypsin-EDTA to the cells. Carefully monitor the digestion under an inverted microscope.The outer layer of myoepithelial cells will begin to detach within three to six minutes. When the myoepithelial cells have detached, transfer the supernatant into a 15 milliliter tube containing two milliliters of 10%FBS in DPBS. Without disturbing the luminal epithelial cells, gently rinse the dish with two milliliters of DPBS before adding a fresh milliliter of trypsin-EDTA to the remaining cells.After seven to 15 minutes, quench the enzymatic reaction with two milliliters of 10%FBS in PBS and transfer the cells to a new 15 milliliter tube. It's critical to closely monitor the cells during the differential trypsinization and to not over digest the cells. When both fractions have been collected, centrifuge the cells to remove any residual dissociation reagent and resuspend the pellets in 250 microliters of maintenance medium per tube.After counting, dilute each cell population to a 1.2 times 10 to the four cells per well concentration in fresh maintenance medium. Add 90 microliters of 50%extracellular matrix in DMEM-F12 without phenol red to each well of an eight-well chamber slide. When all of the wells have been coated, solidify the base layer in the cell culture incubator for 30 minutes.During this polymerization, collect the cell fractions by centrifugation and resuspend each population in 100 microliters of 10%extracellular matrix in 90%growth medium per well. Next, add 100 microliters of each cell suspension to each well and allow the co-cultures to settle for 20 minutes in the cell culture incubator. At the end of the incubation, carefully add 100 microliters of growth medium down the side of each chamber wall and return the slide to the cell culture incubator.Image the cells every 24 hours to track their growth and gently renew the growth medium every two to three days. To fix the organoids for immunostaining, at the appropriate experimental time point, carefully aspirate the medium from each slide to be imaged and rinse each well with 200 microliters of DPBS per well. Fix the organoids with 200 microliters of four degrees Celsius paraformaldehyde for 10 minutes at room temperature before treating the organoids with 200 microliters of 0.2%glycine in DPBS per well for 30 minutes at room temperature.At the end of the incubation, permeabilize the organoids with 0.25%triton X-100 in DPBS for 10 minutes at room temperature followed by the blocking of nonspecific binding with 5%donkey serum or the appropriate serum that matches the species of the secondary antibody in DPBS for one hour with rocking. Then label the cells with 125 to 200 microliters of the appropriate primary antibodies of interest in 1%donkey serum in DPBS overnight at four degrees Celsius with rocking. The next morning, wash each well two times with 200 microliters of DPBS plus triton X-100 before labeling the organoids with the appropriate fluorescence conjugated secondary antibodies for 45 minutes at room temperature with rocking protected from light.Then wash each well two times with fresh DPBS plus triton X-100 as demonstrated and image the organoids by fluorescence microscopy according to standard protocols. After 24 hours of incubation, purified epithelial fragments adhered to the bottom of the culture dish forming flat pancake-like structures with an outer layer of myoepithelial cells encircling an inner layer of luminal epithelial cells. Trypsin treatment differentially detaches the cells with the myoepithelial cells detaching first and appearing as bright rounded cells that encircle the core of remaining cuboidal luminal epithelial cells.The overall purity of the two cell compartments is about 90%as assessed by the keratin-14 and E-cadherin expression within the two populations. After 10 days of culture in 10%extracellular matrix on a 50%extracellular matrix base as demonstrated, the myoepithelial luminal epithelial cell organoids formed large branched structures with well-developed lumens. Differentiation at day five with the addition to alveologenesis medium induces the formation of larger, more branched milk-containing organoids.It is important to remember to wash the strainer carefully when collecting the epithelium to ensure that there are no stromal contaminants. To query the changes in gene expression, researchers can collect RNA from the organoids by releasing them from the extracellular matrix using a recovery solution. Paraformaldehyde is considered hazardous and researchers should use personal protective equipment and work inside a fume hood when using this reagent.

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