This study was supported by funding provided by METAvivor, the Peter Carlson Trust, Theresa&#39;s Research Foundation, and the NCI/UTSW Simmons Cancer Center P30 CA142543. We acknowledge the assistance of the University of Texas Southwestern Tissue Management Shared Resource, a shared resource at the Simmons Comprehensive Cancer Center, which is supported in part by the National Cancer Institute under award number P30 CA142543. Special thanks to all members of the Chan Lab.

All mouse tissue utilized in this manuscript has been ethically collected in accordance with the Institutional Animal Care and Use Committee (IACUC) regulations and guidelines of the University of Texas Southwestern Medical Center. Likewise, all the patients consented prior to tissue donation under the oversight of an Institutional Review Board (IRB), and the samples were deidentified.
NOTE: This protocol describes the generation of organoids from primary tissue.
1. Overnight preparation of materials
<ol>
	<li>For embedding in basement extracellular matrix (BECM), thaw the BECM aliquot by leaving it at 4 &#176;C.</li>
</ol>
2. Preparing collagenase and bovine serum albumin (BSA) coating solution
<ol>
	<li>Prepare 2.64% BSA/PBS coating solution (BSA Solution): Sterile filter 50 mL of phosphate-buffered saline (PBS) and 4.10 mL of 30% BSA solution using a 50 mL/0.2 &#181;m filter flask. This BSA solution can be filtered and used again.</li>
	<li>Prepare collagenase solution for mouse mammary tissue: Sterile filter 27 mL of basal cell medium, 1.5 mL of fetal bovine serum (FBS), 30 &#181;L of 50 mg/mL gentamicin, 15 &#181;L of 10 mg/mL insulin, 600 &#181;L of 0.1 g/mL collagenase A, and 600 &#181;L of 0.1 g/mL trypsin using a 50 mL/0.2 &#181;m filter flask. Allocate between 10 mL and 30 mL of collagenase solution per mouse, depending on tissue mass.</li>
	<li>Prepare collagenase solution for human breast tissue: Sterile filter 18 mL of RPMI-1640, 200 &#181;L of 100x penicillin-streptomycin solution (pen/strep), 200 &#181;L of 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) Buffer, 1 mL of FBS, and 400 &#181;L of 0.1 g/mL Collagenase A using a 50 mL/0.2 &#181;m filter flask. Allocate between 10 mL and 20 mL per tissue sample, depending on the tissue mass.</li>
</ol>
3. Preparing media
<ol>
	<li>Prepare organoid media: Sterile filter basal cell medium with 1% pen/strep, and 1% Insulin-Transferrin-Selenium (ITS) using a 50 mL/0.2 &#181;m filter flask. To make organoid growth factor media, add basic fibroblast growth factor (bFGF) to a final concentration of 2.5 nM.</li>
	<li>Prepare mammary epithelial media: To make 100 mL of mammary epithelial media, sterile filter high-glucose common basal medium with 1 mL of 100x glutamine supplement, 1 mL of pen/strep, 1 mL of 10 mM HEPES buffer (7.3 pH), 250 &#181;L of 30% BSA stock, 1 &#181;L of 1 mg/mL cholera toxin stock, 1 mL of 50 &#181;g/mL hydrocortisone stock in PBS, 50 &#181;L of 10 mg/mL human insulin solution, 10 &#181;L of 50 &#181;g/mL epidermal growth factor (EGF) stock, and 1% FBS using a 150 mL/0.2 &#181;m filter flask. Store the media at 4 &#176;C and use for up to 2 weeks.</li>
	<li>Prepare amphotericin wash: Sterile filter PBS with 2% pen/strep and 2% amphotericin B. Store at 4 &#176;C.</li>
</ol>
4. Collecting and digesting tissue
<ol>
	<li>Mouse mammary tissue
	<ol>
		<li>Euthanize the mouse by placing in a CO2 chamber for 2-5 min, and then follow with cervical dislocation. With the ventral side facing upward, splay mouse limbs and use four 19 G needles to pin the mouse by the paws to a board covered with an absorbent pad. Spray with 70% ethanol to smooth the fur down and sanitize the skin. Wipe away feces with a gauze pad or tissue.</li>
		<li>Starting just above the anogenital region, cut upward from the midline using surgical scissors, taking care not to pierce through the peritoneum. Upon reaching the chin, make lateral cuts down both clavicles and down hind legs, and pin the skin of the mouse taut to the board to expose mammary fat pads.</li>
		<li>Locate the inguinal mammary fat pads on wild-type mice by identifying the inguinal lymph node located near the hindlimbs. Locate the thoracic mammary fat pads on wild-type mice as the thicker, vascularized tissue beneath the front limbs.</li>
		<li>Use forceps to elevate the mammary fat pad. Avoid collecting underlying muscle. Using the blunt end of sharp-blunt scissors, create a pocket underneath the mammary fat pad, away from the skin. Cut away the mammary fat pad in one complete piece.</li>
		<li>Once mammary fat pads have been removed, rinse in PBS before placing them in a sterile tissue culture dish. Quickly transfer to a tissue culture hood. 
		NOTE: For mammary tumors, use a cotton swab wetted with PBS to gently roll the tumor away from the skin. Be certain to avoid darkened or soft areas, as dark discoloration indicates necrosis, which will not yield healthy organoids.</li>
		<li>Mince the mammary tumors with a #10 or #11 scalpel to loosen tissue until it reaches a paste-like consistency. Transfer the minced tissue into a conical tube containing 10-30 mL of collagenase solution using a scalpel. To ensure all tissue is collected, pipette 1 mL of collagenase solution onto the tissue culture plate and back into the conical tube. 
		NOTE: To produce larger organoids, mechanical digestion should be minimized, leaving some small visible pieces of tissue intact. Alternatively, tissue can be stored frozen for later organoid preparation with minimal loss in viability. To do so, wash tissue in a solution of PBS or basal cell media + 1% FBS, and then coarsely mince to increase surface area (keeping the tissue in one or two solid pieces). Move the tissue to a cryo-vial, top with freezing media (90% FBS + 10% dimethyl sulfoxide (DMSO)). Freeze the vials in a controlled-rate freezing container overnight before moving to storage in liquid nitrogen.</li>
		<li>Place the conical tube into a 37 &#176;C benchtop shaking incubator at 180 RPM until the tissue becomes stringy and the collagenase solution becomes cloudy. For example, a large tissue mass (around 500-800 mg) from a tumor takes 30-60 min. A smaller tissue mass (around 100-300 mg) from wild-type mammary fat pads takes about 20-30 min. If uncertain, check at 5 min intervals.</li>
		<li>After incubation, centrifuge the conical tube for 10 min at 550 x g at room temperature (RT).</li>
		<li>Carefully aspirate the supernatant from the conical tube. 
		NOTE: Moving forward, pre-coat all pipette tips, serological pipets, and conical tubes with BSA solution to avoid loss of organoids due to adhesion to plastic.</li>
		<li>Add 8 mL of basal cell media and 80 &#181;L of DNase solution [2 U/&#181;L] to the tube. Invert gently for 1-3 min.</li>
		<li>Add 12 mL of basal cell media to the tube and carefully mix by pipetting or gently rotate the tube 15 times to mix.</li>
		<li>Centrifuge for 10 min at 550 x g at RT.</li>
		<li>Aspirate the supernatant, and then resuspend the pellet in 12 mL of basal cell media.</li>
		<li>Let the heaviest tissue fragments settle to the bottom. Collect the supernatant with a serological pipet and transfer to a 15 mL conical tube. This suspension contains epithelial organoids and stromal/immune cells.</li>
	</ol>
	</li>
	<li>Human breast tissue
	<ol>
		<li>Process the human breast tissue samples for organoids or store them within 24 h of collection. Store the samples at 4 &#176;C in CO2-Independent media with 1% 100x antibiotic-antimycotic.</li>
		<li>Transfer the tissue into a tissue culture plate. Tilt the plate and aspirate the excess storage media, and then rinse the tissue with 5 mL of the amphotericin B wash. Remove the amphotericin B wash immediately.</li>
		<li>Mince the tissue sample with a #10 or #11 scalpel to loosen the tissue until it reaches a paste-like consistency. Transfer the minced tissue into a conical tube containing 10-30 mL of collagenase solution using a scalpel. To ensure all tissue is collected, pipette 1 mL of collagenase solution onto the tissue culture plate and return to the conical tube. 
		NOTE: To produce larger organoids, mechanical digestion should be minimized, leaving some small visible pieces of tissue intact. Starting tissue mass for this protocol ranged between 100-250 mg. Alternatively, tissue can be stored frozen for later organoid preparation with minimal loss in viability after step 4.2.2 in 90% FBS + 10% DMSO as above.</li>
		<li>Place the conical tube into a 37 &#176;C benchtop shaking incubator at 180 RPM until the tissue becomes smaller and collagenase becomes cloudy. Check the tissue at 5 min intervals. Collagenase dissociation of human samples takes roughly 5-20 min.</li>
		<li>After incubation, spin down the conical tube for 10 min at 550 x g at RT.</li>
		<li>Carefully aspirate the supernatant from the conical tube. 
		NOTE: Moving forward, pre-coat all the pipette tips, serological pipets, and conical tubes with BSA solution to avoid loss of organoids due to adhesion to plastic.</li>
		<li>Add 4 mL of basal cell media and 40 &#181;L of DNase solution [2 U/&#181;L] to the conical tube. Invert gently for 3 min.</li>
		<li>Add 6 mL of basal cell media to the tube and carefully mix by pipetting or gently rotate the conical tube 15 times to mix.</li>
		<li>Centrifuge for 10 min at 550 x g at RT.</li>
		<li>Aspirate the supernatant, and then resuspend the pellet in 10 mL of basal cell media.</li>
		<li>Let the heaviest tissue fragments settle to the bottom. Collect the supernatant and transfer it to a 15 mL conical tube. This suspension contains epithelial organoids and stromal/immune cells.</li>
	</ol>
	</li>
</ol>
5. Differential centrifugation
<ol>
	<li>Pulse to 550 x g for 3-4 s at RT, aspirate the supernatant, and resuspend the pellet in 10 mL of basal cell media. Repeat this step three more times (a total of four spins).</li>
	<li>After each centrifugation, ensure that the pellet becomes increasingly opaque. This remaining pellet will be epithelial organoids.</li>
</ol>
6. Collecting small organoids
<ol>
	<li>Pulse to 80-100 x g for 3 s at RT. Collect the supernatant into a fresh BSA-coated tube, and then pulse to 550 x g for 3 s at RT to pellet the small organoids.</li>
</ol>
7. Embedding organoids in BECM
<ol>
	<li>Aliquot appropriate suspension of organoids into microcentrifuge tubes according to the organoid density relative to the amount of BECM per well (Table 1). For example, use 50-100 organoids for 20 &#181;L of BECM in one well of a 96-well plate. 
	NOTE: Organoid density can be counted using the following formula: ((Average number of organoids)/50 &#181;L) x dilution factor.</li>
	<li>Perform all the steps on ice. Place the thawed BECM on ice in a tissue culture hood. While preparing the hood, place all the pipette tips on ice to cool.</li>
	<li>Centrifuge the microcentrifuge tubes for 10 min at 300 x g at RT. Discard the supernatant from the tubes. Move the tubes with organoid pellets to ice and add the appropriate volume of BECM to each microcentrifuge tube (Table 1). 
	NOTE: Since BECM is viscous, add in additional volumes (approximately 10%-20% extra of dome volume) of BECM to account for the loss in the pipet tip.</li>
	<li>Gently pipette up and down to resuspend the organoids in BECM, taking care not to produce bubbles.</li>
	<li>Slowly and carefully pipette the BECM-suspended organoids onto the plating surface. While pipetting, slowly bring up the pipette to create a dome. Fill all the empty wells with PBS to maintain humidity.</li>
	<li>Place the plate into a 37 &#176;C incubator for 1 h to allow BECM to solidify, and then cover with the appropriate volume of media (Table 1).</li>
</ol>
8. Embedding organoids in collagen
<ol>
	<li>Perform all the steps on ice. Prepare the collagen solution by combining, in sequential order, 375 &#181;L of 10x DMEM, 100 &#181;L of 1 N sodium hydroxide (NaOH), and 3 mL of rat tail collagen I solution in a 15 mL conical tube. Pipette mix, taking care not to make bubbles. Titrate the solution to a pH of 7.2-7.4 with small amounts (increments of 1-3 &#181;L) of NaOH or 10x DMEM as needed.</li>
	<li>Coat the bottom of the wells with the minimum amount of collagen required to fully cover the bottom of the well. One approach is to place a small amount of collagen and rock the plate from side to side to coat. Allow the collagen underlays to set at 37 &#176;C for 30 min-2 h.</li>
	<li>Aliquot the appropriate suspension of organoids into microcentrifuge tubes according to the organoid density relative to the amount of collagen per well (Table 1). Place in a 37 &#176;C incubator.</li>
	<li>Store the collagen solution at 4 &#176;C and monitor polymerization by checking for fiber formation under a microscope every 10 min. Collagen will reach appropriate polymerization between 30 min-2 h. 
	NOTE: Proper polymerization can be determined by the presence of fibers branching throughout the matrix. Proceed to step 8.5 immediately once a few fibers are observed in the field of view.</li>
	<li>Centrifuge the microcentrifuge tubes at 300 x g for 10 min&#160;at RT. Discard the supernatant from the tubes. Move the organoid pellets to ice and add the appropriate volume of collagen to each microcentrifuge tube (Table 1). 
	NOTE: Since collagen is viscous, add in additional volumes (approximately 10%-20% extra of dome volume) of collagen to account for the loss in the pipet tip.</li>
	<li>Gently pipette up and down to resuspend organoids in collagen, taking care not to produce bubbles.</li>
	<li>Slowly and carefully pipette the collagen-suspended organoids onto the plating surface. While pipetting, slowly bring up the pipette to create a dome. Fill all the empty wells with PBS to maintain humidity.</li>
	<li>Place the plate into a 37 &#176;C incubator for 1 h to allow collagen to solidify, and then cover with the appropriate volume of media (Table 1).</li>
	<li>NOTE : Organoids maintain viability in 3D matrix for up to 7 days. If imaging is being used for analysis, proceed to steps 9 and 10.</li>
</ol>
9. Fixing embedded organoids
<ol>
	<li>Use a pipette to remove all media from the wells without making contact with ECM domes.</li>
	<li>Fix with 4% paraformaldehyde (PFA)/PBS solution for 5 min.</li>
	<li>Wash two to three times by applying PBS to wells after placing them on a slowly moving shaker. After washes, apply fresh PBS to the domes and store the plate at 4 &#176;C.</li>
</ol>
10. Immunofluorescent staining of embedded organoids
<ol>
	<li>Preparing solutions for immunofluorescent staining
	<ol>
		<li>Permeabilization buffer: Prepare a PBS solution with 10% Triton X-100.</li>
		<li>Blocking buffer: Prepare a PBS solution with 10% FBS and 0.2% Triton X-100.</li>
		<li>Antibody dilution buffer: Prepare a PBS solution with 2% FBS and 0.2% Triton X-100.</li>
	</ol>
	</li>
	<li>Permeabilization and blocking
	<ol>
		<li>Pipette enough permeabilization buffer to cover the ECM domes. Incubate for 1 h at RT on a slowly moving shaker.</li>
		<li>Remove the permeabilization buffer with a pipette and apply the same volume of blocking buffer for 3 h.</li>
	</ol>
	</li>
	<li>Primary antibody incubation
	<ol>
		<li>Remove the blocking buffer with a pipette and apply the antibody dilution buffer containing the primary antibody at manufacturer-specified concentrations. Incubate the sample at 4 &#176;C for 12-16 h on a slowly-moving shaker.</li>
		<li>Remove the primary antibody solution and wash the samples three times for 10 min with PBS at RT on a slowly moving shaker.</li>
	</ol>
	</li>
	<li>Secondary antibody incubation and nuclear stain
	<ol>
		<li>Aspirate the remaining PBS wash and apply the antibody dilution buffer containing secondary antibody at manufacturer-specified concentrations. Additionally, add DAPI or Hoechst (to label nuclei) or phalloidin (to label actin) to the buffer at a 1:250 ratio. Incubate for 2-4 h at RT on a slowly-moving shaker.</li>
		<li>Remove the secondary antibody solution and wash samples three times for 10 min in PBS at RT on a shaker. Store the samples in PBS at 4 &#176;C until imaging.</li>
	</ol>
	</li>
</ol>

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T., Wolfe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+processing+and+hematoxylin+and+eosin+staining.">Tissue processing and hematoxylin and eosin staining.</a> Methods in Molecular Biology. 1180, 31-43 (2014).</li><li>LeSavage, B. L., Suhar, R. A., Broguiere, N., Lutolf, M. P., Heilshorn, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+cancer+organoids.">Next-generation cancer organoids.</a> Nature Materials. 21 (2), 143-159 (2022).</li><li>Nguyen-Ngoc, K. V., 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=3D+culture+assays+of+murine+mammary+branching+morphogenesis+and+epithelial+invasion.">3D culture assays of murine mammary branching morphogenesis and epithelial invasion.</a> Methods in Molecular Biology. 1189, 135-162 (2015).</li><li>Padmanaban, V., 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=Organotypic+culture+assays+for+murine+and+human+primary+and+metastatic-site+tumors.">Organotypic culture assays for murine and human primary and metastatic-site tumors.</a> Nature Protocols. 15 (8), 2413-2442 (2020).</li></ol>

The authors declare no conflicts of interest.

Different methods have been described in the literature to generate tumor organoids. This protocol highlights a method for generating tumor organoids directly from the tumor without passaging. Using this method, tumor organoids are producible within hours of initiating the procedure and generate close to 100% viable organoids compared to 70% reported in the literature31. In comparison, other methods require serial passaging of cells into organoids over several weeks. Thus, the downstream applicati...

The ability to model epithelial cells in vitro has been the foundation of modern biomedical research because it captures cellular features that are not accessible in vivo. For instance, growing epithelial cell lines in a two-dimensional plane can provide an assessment of the molecular changes that occur in an epithelial cell during proliferation1. Furthermore, measuring the dynamic regulation between signaling and gene expression is limited in in vivo systems2. In cancer research, cancer epithelial cell line modeling has enabled the identification of molecular drivers of disease progression and potential drug targets3. However, growing cancer epithelial cell lines on a two-dimensional plane has limitations, as most are genetically immortalized and modified, often clonal in nature, selected for their ability to grow in non-physiologic conditions, limited in their assessment of three-dimensional (3D) tumor tissue architecture, and do not adequately model microenvironment interactions within a realistic tissue environment4. These constraints are particularly evident in modeling metastasis, which in vivo includes several distinct biological stages, including invasion, dissemination, circulation, and colonization at the distant organ site5.
Cancer epithelial organoids have been developed to better recapitulate the 3D environment and behavior of tumors6,7,8. Organoids were first developed from single LRG5+ intestinal crypt cells and differentiated to represent the 3D structure of crypt-villus units that maintained the hierarchical structure of the small intestine in vitro9. This approach permitted real-time visualization and characterization of self-organizing tissue architecture under homeostatic and stress conditions. As a natural extension, cancer epithelial organoids were developed to model many different cancer types, including colorectal10, pancreatic11, breast12, liver13, lung14, brain15, and gastric cancers16. Cancer epithelial organoids have been exploited to characterize cancer evolution17,18 and metastatic spatiotemporal behaviors19,20 and interrogate tumor heterogeneity21, and test chemotherapies22. Cancer epithelial organoids have also been isolated and collected during ongoing clinical trials to predict patient response to anticancer agents and radiation therapy ex vivo8,23,24,25. Furthermore, systems incorporating cancer epithelial organoids can be combined with other non-cancer cells, such as immune cells, to form a more comprehensive model of the tumor microenvironment to visualize interactions in real-time, uncover how cancer epithelial cells change the fundamental nature of cytotoxic effector immune cells such as natural killer cells, and test potential immunotherapies and antibody-drug dependent cytotoxic activity26,27,28. This article demonstrates a method of generating epithelial organoids without passaging and embedding in collagen and basement extracellular matrix (ECM). Additionally, techniques for downstream imaging of isolated organoids are also shared.

<table><tbody><tr><td>10 mM HEPES Buffer</td><td>Gibco&amp;nbsp;</td><td>15630080</td><td></td></tr><tr><td>100x Antibiotic-Antimycotic&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>15240-096</td><td></td></tr><tr><td>100x Glutamax</td><td>Life Technologies&amp;nbsp;</td><td>35050-061</td><td>Glutamine supplement</td></tr><tr><td>100x Insulin-Transferrin-Selenium (ITS)&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>51500-056</td><td></td></tr><tr><td>100x Penicillin/Streptomycin (Pen/Strep)</td><td>Sigma&amp;nbsp;</td><td>P4333</td><td></td></tr><tr><td>10x DMEM</td><td>Sigma&amp;nbsp;</td><td>D2429</td><td></td></tr><tr><td>50 mL/0.2 &amp;micro;m filter flask</td><td>Fisher&amp;nbsp;</td><td>#564-0020</td><td></td></tr><tr><td>Amphotericin B</td><td>Life Technologies&amp;nbsp;</td><td>15290-018</td><td></td></tr><tr><td>bFGF</td><td>Sigma</td><td>F0291</td><td></td></tr><tr><td>BSA Solution (32%)</td><td>Sigma&amp;nbsp;</td><td>#A9576</td><td></td></tr><tr><td>Cholera Toxin&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>C8052</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt;-Independent Medium&amp;nbsp;</td><td>Gibco</td><td>18045-088</td><td></td></tr><tr><td>Collagenase A&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>C2139</td><td></td></tr><tr><td>Deoxyribonuclease I from bovine pancreas (DNase)</td><td>Sigma</td><td>D4263</td><td></td></tr><tr><td>DMEM with 4500 mg/L glucose, sodium pyruvate, and sodium bicarbonate, without L-glutamine, liquid, sterile-filtered, suitable for cell culture</td><td>Sigma</td><td>D6546</td><td>Common basal medium</td></tr><tr><td>D-MEM/F12&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>#10565-018</td><td>Basal cell medium</td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (D-PBS)&amp;nbsp;</td><td>Sigma</td><td>#D8662</td><td>PBS</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Sigma&amp;nbsp;</td><td>#F0926</td><td></td></tr><tr><td>Gentamicin&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>#15750-060</td><td></td></tr><tr><td>Human epidermal growth factor (EGF)</td><td>Sigma&amp;nbsp;</td><td>E9644</td><td></td></tr><tr><td>Hydrocortisone&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>H0396</td><td></td></tr><tr><td>Insulin&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>#I9278</td><td></td></tr><tr><td>Matrigel&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>#354230</td><td>Basement Extracellular Matrix (BECM)</td></tr><tr><td>NaOH (1 N)</td><td>Sigma&amp;nbsp;</td><td>S2770</td><td></td></tr><tr><td>Rat Tail Collagen I</td><td>Corning&amp;nbsp;</td><td>354236</td><td></td></tr><tr><td>RPMI-1640 media</td><td>Fisher&amp;nbsp;</td><td>SH3002701</td><td></td></tr><tr><td>Trypsin&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>27250-018</td><td></td></tr></tbody></table>

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.

The images featured in Figure 1 provide an example of wild-type and tumorous mammary epithelial organoids from human and mouse tissues. An at-a-glance illustration of the method for isolating epithelial organoids through differential centrifugation is provided in the cartoon workflow in Figure 1A, showing that primary tissues from different species can be processed in near-identical ways while yielding epithelial tissue as shown in the brightfield images (<stron...

Watch this Scientific Journal Video about Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging at JoVE.com

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

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 ...

generating-imaging-mouse-human-epithelial-organoids-from-normal-tumor

Research

JoVE Journal

Cancer Research

2.7K Views.  University of Texas Southwestern Medical Center. Dit protocol is belangrijk voor de productie van geheugenepitheelorganoïden die worden gegenereerd zonder te passeren. Het belangrijkste voordeel van deze techniek is dat organoïden na enzymatische spijsvertering kunnen worden geïsoleerd uit borst- en geheugenweefsel van mens en muis. We voeren een reeks differentiële centrifugatiestappen uit die epitheliale organoïden isoleren zonder cellen te hoeven passeren.Een persoon die deze techniek uitvoert, kan uitdagingen tegenkomen bij ...

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

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) are a rigorous method for studying mammalian development and disease. Our laboratory has developed several GEMMs of prostate cancer (PCa) that lack expression of one or multiple tumor suppressor genes using the site-specific Cre-loxP recombinase system and a prostate-specific promoter. In this article, we describe our method for necropsy of these PCa GEMMs, primarily focusing on dissection of mouse prostate tumors. New methods developed over the last decade have facilitated the culture of epithelial-derived cells to model organ systems in vitro in three dimensions. We also detail a 3D cell culture method to generate tumor organoids from mouse PCa GEMMs. Pre-clinical cancer research has been dominated by 2D cell culture and cell line-derived or patient-derived xenograft models. These methods lack tumor microenvironment, a limitation of using these techniques in pre-clinical studies. GEMMs are more physiologically-relevant for understanding tumorigenesis and cancer progression. Tumor organoid culture is an in vitro model system that recapitulates tumor architecture and cell lineage characteristics. In addition, 3D cell culture methods allow for growth of normal cells for comparison to tumor cell cultures, rarely possible using 2D cell culture techniques. In combination, use of GEMMs and 3D cell culture in pre-clinical studies has the potential to improve our understanding of cancer biology.&#160;

We show a method for necropsy and dissection of mouse prostate cancer models, focusing on prostate tumor dissection. A step-by-step protocol for generation of mouse prostate tumor organoids is also presented.

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...

Genetics

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 ...

Medicine

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

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 ...

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 ...

Generation of Multicellular Human Primary Endometrial Organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids.

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...

Developmental Biology

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 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 ...

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

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 ...

Biology

Generation and Characterization of Murine Oral Mucosal Organoid Cultures

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, gingiva, lips, palate, and tongue. Its uppermost layer, the oral epithelium, is maintained by adult stem cells throughout life. Proliferation and differentiation of adult epithelial stem cells have been intensively studied using in vivo mouse models as well as two-dimensional (2D) feeder-cell based in vitro models. Complementary to these methods is organoid technology, where adult stem cells are embedded into an extracellular matrix (ECM)-rich hydrogel and provided with a culture medium containing a defined cocktail of growth factors. Under these conditions, adult stem cells proliferate and spontaneously form three-dimensional (3D) cell clusters, the so-called organoids. Organoid cultures were initially established from murine small intestinal epithelial stem cells. However, the method has since been adapted for other epithelial stem cell types. Here, we describe a protocol for the generation and characterization of murine oral mucosal organoid cultures. Primary epithelial cells are isolated from murine tongue tissue, embedded into an ECM hydrogel, and cultured in a medium containing: epidermal growth factor (EGF), R-spondin, and fibroblast growth factor (FGF) 10. Within 7 to 14 days of initial seeding, the resulting organoids can be passaged for further expansion and cryopreservation. We additionally present strategies for the characterization of established organoid cultures via 3D whole-mount imaging and gene-expression analysis. This protocol may serve as a tool to investigate oral epithelial stem cell behavior ex vivo in a reductionist manner.

We present a method for the generation and characterization of oral mucosal organoid cultures derived from the tongue epithelium of adult mice.

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, ...

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

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