Name: generating and imaging mouse and human epithelial organoids from normal and tumor mammary tissue without passaging
Uploaded: 2022-11-11T14:30:00
Description: 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

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.
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<ol>
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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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		<tr>
			<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 ...

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