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.
<str...

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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.

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			<td>10 mM HEPES Buffer</td>
			<td>Gibco&#160;</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>100x Antibiotic-Antimycotic&#160;</td>
			<td>Gibco&#160;</td>
			<td>15240-096</td>
			<td></td>
		</tr>
		<tr>
			<td>100x Glutamax</td>
			<td>Life Technologies&#160;</td>
			<td>35050-061</td>
			<td>Glutamine supplement</td>
		</tr>
		<tr>
			<td>100x Insulin-Transferrin-Selenium (ITS)&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>51500-056</td>
			<td></td>
		</tr>
		<tr>
			<td>100x Penicillin/Streptomycin (Pen/Strep)</td>
			<td>Sigma&#160;</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>10x DMEM</td>
			<td>Sigma&#160;</td>
			<td>D2429</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL/0.2 &#181;m filter flask</td>
			<td>Fisher&#160;</td>
			<td>#564-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Life Technologies&#160;</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&#160;</td>
			<td>#A9576</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera Toxin&#160;</td>
			<td>Sigma&#160;</td>
			<td>C8052</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2-Independent Medium&#160;</td>
			<td>Gibco</td>
			<td>18045-088</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase A&#160;</td>
			<td>Sigma&#160;</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&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>#10565-018</td>
			<td>Basal cell medium</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (D-PBS)&#160;</td>
			<td>Sigma</td>
			<td>#D8662</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma&#160;</td>
			<td>#F0926</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>#15750-060</td>
			<td></td>
		</tr>
		<tr>
			<td>Human epidermal growth factor (EGF)</td>
			<td>Sigma&#160;</td>
			<td>E9644</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone&#160;</td>
			<td>Sigma&#160;</td>
			<td>H0396</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin&#160;</td>
			<td>Sigma&#160;</td>
			<td>#I9278</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel&#160;</td>
			<td>Corning&#160;</td>
			<td>#354230</td>
			<td>Basement Extracellular Matrix (BECM)</td>
		</tr>
		<tr>
			<td>NaOH (1 N)</td>
			<td>Sigma&#160;</td>
			<td>S2770</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat Tail Collagen I</td>
			<td>Corning&#160;</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 media</td>
			<td>Fisher&#160;</td>
			<td>SH3002701</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin&#160;</td>
			<td>Life Technologies&#160;</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 ...

This protocol is significant for the production of memory epithelial organoids that are generated without passaging. The main advantage of this technique is that organoids can be isolated from human and mouse breast and memory tissue after enzymatic digestion. We perform a series of differential centrifugation steps that isolate epithelial organoids without having to passage cells.An individual performing this technique may face challenges when embedding the tissues in collagen or matrix and plating them within a 96-well plate. Furthermore, using properly polymerized collagen and plating stable domes is the key to seeing invasion. To begin, collect the mammary tissue of a euthanized mouse by splaying mouse limbs and using four 19 gauge needles, pin the mouse by the paws to a board covered with an absorbent pad with the ventral side facing upward.Spray with 70%ethanol to smooth the fur down and sanitize the skin and wipe away feces with a gauze pad or tissue. 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 the hind legs and pin the skin of the mouse taut to the board to expose mammary fat pads.After locating the inguinal and thoracic mammary fat pads on wild type mice, use forceps to elevate the mammary fat pad. Using the blunt end of sharp blunt scissors, create a pocket underneath the mammary fat pad away from the skin and cut away the mammary fat pad in one complete piece. Once mammary fat pads have been removed, rinse in PBS before placing them in a sterile tissue culture dish and then quickly transfer to a tissue culture hood.Mince the mammary tumors with a number 10 or number 11 scalpel to loosen tissue until it reaches a paste-like consistency. Transfer the minced tissue into a conical tube containing 10 to 30 milliliters of collagenase solution using a scalpel. To ensure all tissue is collected, pipette one milliliter of collagenase solution onto the tissue culture plate and back into the conical tube.Place the conical tube into a benchtop shaking incubator until the tissue becomes stringy and the collagenase solution becomes cloudy. Spin down 50 milliliters of solution for five to 10 minutes at 1, 500 RPM. Aspirate supernatant, and add 12 milliliters of the basal medium and mix by pipetting up and down three to four times or gently rotate the wrist while holding the tube 15 times.Again, spin down aspirate supernatant, add basal medium, and mix by pipetting or tube rotation as demonstrated earlier. Once the heaviest tissue fragments settle to the bottom collect the supernatant with a serological pipette and transfer it to a 15 milliliter conical tube. After each centrifugation, ensure that the pellet becomes increasingly opaque.Aliquot appropriate suspension of organoids into microcentrifuge tubes according to the organoid density relative to the amount of BECM per well. Centrifuge the microcentrifuge tubes for 10 minutes at 300 G at room temperature and 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.Gently pipette up and down to resuspend the organoids in BECM, taking care not to produce bubbles. Slowly and carefully pipette the BECM suspended organoids onto the plating surface and fill all the empty wells with PBS to maintain humidity. Place the plate into a 37 degrees Celsius incubator for one hour to allow BECM to solidify and then cover with the appropriate volume of media.To prepare the collagen solution, combine 375 microliters of 10 times the concentration of DMEM, 100 microliters of one normal sodium hydroxide, and three milliliters of rat tail collagen I solution in a 15 milliliter conical tube. Pipette mix, taking care not to make bubbles, and titrate the solution to a pH of 7.2 to 7.4 with a small amount of sodium hydroxide or 10 times the concentration of DMEM as needed. Coat the bottom of the wells with the minimum amount of collagen required to fully cover the bottom of the well by placing a small amount of collagen and rock the plate from side to side to coat.Allow the collagen underlays to set at 37 degrees Celsius for 30 minutes to two hours. Aliquot the appropriate suspension of organoids into microcentrifuge tubes according to the organoid density relative to the amount of collagen per well. Store the collagen solution at four degrees Celsius and monitor polymerization by checking for fiber formation under a microscope every 10 minutes.Centrifuge the microcentrifuge tubes at 300 G for 10 minutes at room temperature and discard the supernatant from the tubes. Move the organoid pellets to ice and add the appropriate volume of collagen to each microcentrifuge tube. Gently pipette up and down to resuspend organoids in collagen, taking care not to produce bubbles.Slowly and carefully pipette the collagen suspended organoids onto the plating surface. The immunofluorescent images of embedded organoids in either basement extracellular matrix or collagen were obtained to study the inter-species tissue composition similarities. Organoids embedded in collagen matrix can be used for an invasion assay and analyzed by tracking the expansion of tendrils branching out from the organoid itself either through membrane tomato labeling or phalloidin staining of actin.Lastly, hematoxylin and eosin staining of paraffin-embedded organoids show that organoids maintain the same histology of breast cancer. It is crucial to keep BECM and collagen cold while pipetting gelled domes to avoid premature polymerization. Additionally, prolonged fixing of domes in PFA will lead to gelled solution if performing immunofluorescent staining.Organoids can also be used in in vitro and in vivo procedures such as drug screens and mouse models of metastasis. These methods can provide insights into therapeutic sensitivity of tumor tissue, metastatic properties, and immune interactions.

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Research

JoVE Journal

Cancer Research

2.6K visualizações.  University of Texas Southwestern Medical Center. Este protocolo é significativo para a produção de organoides epiteliais de memória que são gerados sem passar. A principal vantagem desta técnica é que os organoides podem ser isolados do tecido mamário e de memória humana e de camundongo após a digestão enzimática. Realizamos uma série de etapas de centrifugação diferencial que isolam organoides epiteliais sem ter que passar células.Um indivíduo que realiza esta técnica pode enfrentar desafios ao incorporar os tecido...