Name: isolation and characterization of patient-derived pancreatic ductal adenocarcinoma organoid models
Uploaded: 2020-01-14T13:00:00
Description: Watch this Scientific Journal Video about Isolation and Characterization of Patient-derived Pancreatic Ductal Adenocarcinoma Organoid Models at JoVE.com

We are grateful for the support of the UC San Diego Moores Cancer Center Biorepository and Tissue Technology Shared Resource, members of the Lowy laboratory, and the UC San Diego Department of Surgery, Division of Surgical Oncology. AML is generously supported by NIH CA155620, a SU2C CRUK Lustgarten Foundation Pancreatic Cancer Dream Team Award (SU2C-AACR-DT-20-16), and donors to the Fund to Cure Pancreatic Cancer.

All human tissue collection for research use was reviewed and approved by our Internal Review Board (IRB). All of the following protocols are performed under aseptic conditions in a mammalian tissue culture laboratory environment.
1. Media Preparation
<ol>
	<li>Conditioned media preparation. 
	NOTE: The human pancreatic organoid media requires abundant growth factors and nutrients as well as conditioned media supplementation to provide sufficient growth stimulation for organoid expansio...

<ol>
	<li>Siegel, R. L., Miller, K. D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics,+2018.">Cancer statistics, 2018.</a> CA: A Cancer Journal for Clinicians. 68 (1), 7-30 (2018).</li><li>Khorana, A. A., Mangu, P. B., Katz, M. H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potentially+Curable+Pancreatic+Cancer:+American+Society+of+Clinical+Oncology+Clinical+Practice+Guideline+Update+Summary.">Potentially Curable Pancreatic Cancer: American Society of Clinical Oncology Clinical Practice Guideline Update Summary.</a> Journal of Oncology Practice. 13 (6), 388-391 (2017).</li><li>Winter, J. M., 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=1423+pancreaticoduodenectomies+for+pancreatic+cancer:+A+single-institution+experience.">1423 pancreaticoduodenectomies for pancreatic cancer: A single-institution experience.</a> Journal of Gastrointestinal Surgery. 10 (9), 1210-1211 (2006).</li><li>Von Hoff, D. D., 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=Increased+survival+in+pancreatic+cancer+with+nab-paclitaxel+plus+gemcitabine.">Increased survival in pancreatic cancer with nab-paclitaxel plus gemcitabine.</a> New England Journal of Medicine. 369 (18), 1691-1703 (2013).</li><li>Conroy, T., 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=FOLFIRINOX+versus+gemcitabine+for+metastatic+pancreatic+cancer.">FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer.</a> New England Journal of Medicine. 364 (19), 1817-1825 (2011).</li><li>Baker, L. A., Tiriac, H., Clevers, H., Tuveson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+pancreatic+cancer+with+organoids.">Modeling pancreatic cancer with organoids.</a> Trends in Cancer. 2 (4), 176-190 (2016).</li><li>Sato, T., 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=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459 (7244), 262-265 (2009).</li><li>Boj, S. F., 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=Organoid+models+of+human+and+mouse+ductal+pancreatic+cancer.">Organoid models of human and mouse ductal pancreatic cancer.</a> Cell. 160 (1-2), 324-338 (2015).</li><li>Tiriac, 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=Organoid+Profiling+Identifies+Common+Responders+to+Chemotherapy+in+Pancreatic+Cancer.">Organoid Profiling Identifies Common Responders to Chemotherapy in Pancreatic Cancer.</a> Cancer Discovery. 8 (9), 1112-1129 (2018).</li><li>Tiriac, 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=Successful+creation+of+pancreatic+cancer+organoids+by+means+of+EUS-guided+fine-needle+biopsy+sampling+for+personalized+cancer+treatment.">Successful creation of pancreatic cancer organoids by means of EUS-guided fine-needle biopsy sampling for personalized cancer treatment.</a> Gastrointestinal Endoscopy. , (2018).</li><li>Seino, T., 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=Human+Pancreatic+Tumor+Organoids+Reveal+Loss+of+Stem+Cell+Niche+Factor+Dependence+during+Disease+Progression.">Human Pancreatic Tumor Organoids Reveal Loss of Stem Cell Niche Factor Dependence during Disease Progression.</a> Cell Stem Cell. 22 (3), 454-467 (2018).</li><li>Huang, 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=Ductal+pancreatic+cancer+modeling+and+drug+screening+using+human+pluripotent+stem+cell-+and+patient-derived+tumor+organoids.">Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell- and patient-derived tumor organoids.</a> Nature Medicine. 21 (11), 1364-1371 (2015).</li><li>Walsh, A. J., Castellanos, J. A., Nagathihalli, N. S., Merchant, N. B., Skala, M. 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T., 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=Organoid+Modeling+of+the+Tumor+Immune+Microenvironment.">Organoid Modeling of the Tumor Immune Microenvironment.</a> Cell. 175 (7), 1972-1988 (2018).</li><li>Kopper, O., 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=An+organoid+platform+for+ovarian+cancer+captures+intra-+and+interpatient+heterogeneity.">An organoid platform for ovarian cancer captures intra- and interpatient heterogeneity.</a> Nature Medicine. 25 (5), 838-849 (2019).</li></ol>

Here, we present current protocols for isolating, expanding and characterizing patient-derived PDAC organoids. Our current success rate of establishing organoid culture is over 70%; therefore, these methods have not yet been perfected and are expected to improve and evolve over time. Important consideration should be given to sample size, as PDAC has a low neoplastic cellularity. Consequently, small specimens will contain few tumor cells, and will only generate a handful of organoids. Additionally, many patients receive ...

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease characterized by late diagnosis in most patients, a lack of effective therapies, and a resultant low 5-year overall survival rate that remains less than 10%1. Only 20% of patients are diagnosed with a localized disease suitable for curative surgical intervention2,3. The remaining patients are typically treated with a combination of chemotherapeutic agents that are effective in a minority of patients4,5. To address these pressing clinical needs, researchers are actively working on early detection strategies and the development of more effective therapies. To accelerate clinical translation of important discoveries, scientists are employing genetically engineered mouse models, patient derived xenografts, monolayer cells lines, and, most recently, organoid models6.
Three-dimensional epithelial organoid culture using growth factor and Wnt-ligand rich conditions to stimulate proliferation of untransformed progenitor cells were first described for the mouse intestine7 and were quickly adapted to normal human pancreatic tissue8. In addition to normal ductal tissue, organoid methodology allows for the isolation, expansion, and study of human PDAC8. Importantly, the method supports the establishment of organoids from surgical specimens, as well as fine and core needle biopsies, allowing researchers to study all stages of the disease9,10. Interestingly, patient-derived organoids recapitulate well-described tumor transcriptomic subtypes and may enable development of precision medicine platforms9,11.
Current organoid protocols for PDAC enable the successful expansion of more than 70% of patient samples from chemo-na&iuml;ve patients9. Here we present the standard methods employed by our laboratory to isolate, expand, and characterize patient-derived PDAC organoids. Other PDAC organoid methodologies have been described12,13 but no comparison of these method has been thoroughly performed. As this technology is relatively new and advancing quickly, we expect that these protocols will continue to evolve and improve; however the principles of tissue handling and organoid culture will continue to be useful.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12 channel pipette (p20, p100, or p200) with tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>12 well plates</td>
			<td>Olympus</td>
			<td>25-106</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml LoBind conical tubes</td>
			<td>Eppendorf</td>
			<td>EP0030122208</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml tube Rotator and/or nutator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#176;C CO2 incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#176;C water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>384 well plates</td>
			<td>Corning</td>
			<td>4588</td>
			<td>Ultra low attachment, black and optically clear</td>
		</tr>
		<tr>
			<td>A 83-01</td>
			<td>TOCRIS</td>
			<td>2939</td>
			<td></td>
		</tr>
		<tr>
			<td>ADV DMEM</td>
			<td>ThermoFisher</td>
			<td>12634010</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal-Free Recombinant Human EGF</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated cell counter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>ThermoFisher</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Recovery Solution</td>
			<td>Corning</td>
			<td>354253</td>
			<td>Reagent that depolymerizes the Basement Membrane Extract at 4 &#176;C</td>
		</tr>
		<tr>
			<td>CellTiterGlow</td>
			<td>Promega</td>
			<td>G7570</td>
			<td>Luminescence cell viability reagent</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma</td>
			<td>C2432</td>
			<td></td>
		</tr>
		<tr>
			<td>Computer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CryoStor CS10</td>
			<td>StemCELL Tech</td>
			<td>07930</td>
			<td>Cell Freezing Solution</td>
		</tr>
		<tr>
			<td>Cultrex R-spondin1 (Rspo1) Cells</td>
			<td>Trevigen</td>
			<td>3710-001-K</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>ATCC</td>
			<td>30-2002</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>D5025</td>
			<td></td>
		</tr>
		<tr>
			<td>Drug printer</td>
			<td>Tecan</td>
			<td>D300e</td>
			<td>This is the drug printer we use in our laboratory</td>
		</tr>
		<tr>
			<td>Excel</td>
			<td></td>
			<td></td>
			<td>For data analysis</td>
		</tr>
		<tr>
			<td>Extra Fine Graefe Forceps</td>
			<td>Fine Science Tools</td>
			<td>11150-10</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>ThermoFisher</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>G-418</td>
			<td>ThermoFisher</td>
			<td>10131035</td>
			<td></td>
		</tr>
		<tr>
			<td>Gastrin I (human)</td>
			<td>TOCRIS</td>
			<td>3006</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentle Collagenase/hyaluronidase</td>
			<td>STEMCELL Tech</td>
			<td>7919</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>ThermoFisher</td>
			<td>35050061</td>
			<td>Glutamine solution</td>
		</tr>
		<tr>
			<td>GraphPad Prism</td>
			<td></td>
			<td></td>
			<td>For data analysis</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>ThermoFisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow tissue culture hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-Wnt-3A expressing cells</td>
			<td>ATCC</td>
			<td>CRL-2647</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Tissue Storage Solution</td>
			<td>Miltenyi biotec</td>
			<td>130-100-008</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Matrix</td>
			<td>Corning</td>
			<td>356230</td>
			<td>Basement Membrane Extract (BME), growth factor reduced</td>
		</tr>
		<tr>
			<td>Mr. Frosty Freezing Container</td>
			<td>ThermoFisher</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Acetylcysteine</td>
			<td>Sigma</td>
			<td>A9165</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma</td>
			<td>N0636</td>
			<td></td>
		</tr>
		<tr>
			<td>p1000 pipette with tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>p200 pipette with tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>ThermoFisher</td>
			<td>10010049</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>ThermoFisher</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>primocin</td>
			<td>InvivoGen</td>
			<td>ant-pm-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Rapid-Flow Filter Units (0.2 &#181;m)</td>
			<td>ThermoFisher</td>
			<td>121-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF-10</td>
			<td>Peprotech</td>
			<td>100-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Murine Noggin</td>
			<td>Peprotech</td>
			<td>250-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Disposable Scalpels, #10 Blade</td>
			<td>VWR</td>
			<td>89176-380</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Dishes 10 cm</td>
			<td>Olympus</td>
			<td>25-202</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIZol</td>
			<td>ThermoFisher</td>
			<td>15596018</td>
			<td>Acid Phenol solution</td>
		</tr>
		<tr>
			<td>TrypLE Express</td>
			<td>ThermoFisher</td>
			<td>12605010</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Sigma</td>
			<td>Y0503</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeocin</td>
			<td>ThermoFisher</td>
			<td>R25001</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and characterization of patient-derived pancreatic ductal adenocarcinoma organoid models

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies. Recently, next-generation organoid culture methods enabling the 3-dimensional (3D) modeling of this disease have been described. Patient-derived organoid (PDO) models can be isolated from both surgical specimens as well as small biopsies and form rapidly in culture. Importantly, organoid models preserve the pathogenic genetic alterations detected in the patient&#39;s tumor and are predictive of the patient&#39;s treatment response, thus enabling translational studies. Here, we provide comprehensive protocols for adapting tissue culture workflow to study 3D, matrix embedded, organoid models. We detail methods and considerations for isolating and propagating primary PDAC organoids. Furthermore, we describe how bespoke organoid media is prepared and quality controlled in the laboratory. Finally, we describe assays for downstream characterization of the organoid models such as isolation of nucleic acids (DNA and RNA), and drug testing. Importantly we provide critical considerations for implementing organoid methodology in a research laboratory.

To illustrate the challenges associated with isolating organoids from PDAC, we show the establishment of a patient derived organoid culture from a small hypocellular tumor sample. After initial plating, only a few organoids were visible per well, as shown in Figure 1. Organoids were allowed to grow larger over the span of 2 week and were passaged according to our protocol to establish a more robust culture, as shown in the early and late passage 1 representative pictures (<strong class="xfig...

Watch this Scientific Journal Video about Isolation and Characterization of Patient-derived Pancreatic Ductal Adenocarcinoma Organoid Models at JoVE.com

Isolation and Characterization of Patient-derived Pancreatic Ductal Adenocarcinoma Organoid Models

Patient-derived organoid cultures of pancreatic ductal adenocarcinoma are a rapidly established 3-dimensional model that represent epithelial tumor cell compartments with high fidelity, enabling translational research into this lethal malignancy. Here, we provide detailed methods to establish and propagate organoids as well as to perform relevant biological assays using these models.

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies. Recently, next-generation organoid culture methods enabling the ...

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