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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Imaging+of+Drug-Induced+Metabolism+Changes+in+Murine+and+Human+Pancreatic+Cancer+Organoids+Reveals+Heterogeneous+Drug+Response.">Optical Imaging of Drug-Induced Metabolism Changes in Murine and Human Pancreatic Cancer Organoids Reveals Heterogeneous Drug Response.</a> Pancreas. 45 (6), 863-869 (2016).</li><li>Zhao, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+Reporter+Activity+Assay.">Wnt Reporter Activity Assay.</a> Bio-Protocol. 4 (14), 1183 (2014).</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+or+Gemcitabine+as+Adjuvant+Therapy+for+Pancreatic+Cancer.">FOLFIRINOX or Gemcitabine as Adjuvant Therapy for Pancreatic Cancer.</a> New England Journal of Medicine. 379 (25), 2395-2406 (2018).</li><li>Jimeno, A., 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=A+direct+pancreatic+cancer+xenograft+model+as+a+platform+for+cancer+stem+cell+therapeutic+development.">A direct pancreatic cancer xenograft model as a platform for cancer stem cell therapeutic development.</a> Molecular Cancer Therapeutics. 8 (2), 310-314 (2009).</li><li>Neal, J. 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 ...

This method is significant because patient-derived organoid models are a powerful and rapid tool to study pancreatic cancer as they reflect the genetic and phenotypic heterogeneity of this disease. The main advantage of this technique is that organoids can be isolated with a high success rate from a limited amount of cancer and they can be expanded rapidly in vitro for downstream analysis. Studying organoids'response to cytotoxic drugs may help us understand why tumors become resistant to therapy and to define more effective treatment strategies.To begin this procedure, retrieve the prepared 12-well plate from the incubator. With a sterile cell lifter, gently lift the basement membrane extract dome such that it is floating in the complete media while making sure to avoid scraping the bottom of the well. Using a P1000 pipette, carefully transfer the BME dome and the media to a 15 milliliter conical tube.Repeat this step for every organoid-containing well. Gently wash each well that was harvested with one milliliter of cold basal media and transfer this wash to the tube containing the organoids. Thoroughly mix the solution and organoids with a P1000 pipette and spin down at 200 times g for five minutes.A layer of BME containing organoids in suspension and an organoid pellet will appear at the bottom of the tube. Carefully remove the supernatant making sure to avoid any loss of the BME and organoid layer. Place the tube on ice.Add 10 milliliters of ice cold cell recovery solution to the tube and thoroughly mix by inversion. Incubate on ice for 30 minutes while mixing every three minutes by inversion. After this, centrifuge at 200 times g for five minutes.The organoid pellet should be apparent while the BME layer should be gone. If the BME layer is decreased in size but still visible, incubate for an additional 30 minutes at four degrees Celsius and repeat the centrifugation. Once the BME has been depolymerized, remove the cell recovery solution while leaving behind the organoid pellet.Wash the organoids with 10 milliliters of basal media by mixing with inversion. Centrifuge at 200 times g for five minutes, remove the supernatant, and place the tube with the organoid pellet on ice for at least five minutes. Optionally, if a single cell preparation is desired, add three milliliters of trypsin supplemented with 30 microliters of DNAse I solution to the organoids.Incubate this enzymatic reaction at 37 degrees Celsius with gentle inversion mixing every two minutes for up to 10 minutes. Use a Brightfield microscope to monitor and confirm that the single cell dissociation is successful. To stop the enzymatic reaction, add 10 milliliters of ice cold basal media and spin down at 200 times g for five minutes.Then remove the supernatant. Wash the cells with 10 milliliters of basal media by mixing with gentle inversion. Centrifuge again at 200 times g for five minutes.Remove the supernatant and repeat this wash and centrifuging process once more. Place the tube with the cell pellet on ice for at least five minutes. After this, add ice cold BME to the cell pellet and use a P200 pipette to mix the solution gently until it is homogenous.Then place the tip of the pipette close to the bottom of the tube and pipette up and down at least five to 10 times to mechanically break up the organoids. Use a P200 pipette to spot a 100 microliter dome in the center of a well of a pre-warmed 12-well plate. Repeat until all of the BME solution is dispensed.Then carefully transfer the plate to an incubator at 37 degrees Celsius to allow the BME gel to solidify. After 10 minutes, retrieve the plate and add one milliliter of pre-warmed organoid complete media to each well. Organoid cultures are typically passaged over seven to 10 days depending on the culture density and proliferation.If necessary, top-up the cultures with 200 microliters of pre-warmed organoid complete media every five days to compensate for growth factor depletion and evaporation. After isolating single cells from organoids, resuspend the single cells in one milliliter of human organoid complete media. Next, use an automated cell counter to count the cells making sure to record the cell viability.Calculate the total number of cells and viable cells present in the one milliliter suspension. Then remove the volume equivalent to 400, 000 cells and transfer it to a new tube. Add organoid complete media to this tube to bring the volume up to 7.2 milliliters.For therapeutic testing, prepare cells for plating by mixing 800 microliters of BME with 7.2 milliliters of organoid complete media containing the cells on ice. Transfer the mixture to a reservoir kept on ice and use a 12-channel pipette to plate 20 microliters of this mixture into each well of a 384-well plate resulting in approximately 1, 000 cells being plated per well. Then spin down the plate at 100 times g for one minute in a swing bucket and place the plate into a tissue culture incubator at 37 degrees Celsius.After 24 hours, use a Brightfield microscope to check for the presence of organoids. In this study, patient-derived PDAC organoid are isolated, expanded and characterized. To illustrate the challenges associated with isolating organoids from PDAC, a representative patient-derived organoid culture is established from a small hypocellular tumor sample.Organoids are allowed to grow larger over the span of two weeks and are passaged according to the protocol to establish a more robust culture. Single cells from an established and fast growing representative PDAC organoid are then prepared to demonstrate the outcome of the pharmacotyping protocol. 1, 000 viable cells are plated in each well and allowed to recover over a 24-hour period before the cytotoxic chemotherapeutic agents are dosed.A 9.0 dose assay is performed in triplicate starting with a low dose of 100 picomolar and ending with a high dose of two micromolar. After a five-day treatment, representative pictures are taken for wells treated with the vehicle and with the high dose of each chemotherapeutic agent. Immediately after taking the pictures, cell viability is assessed using luminescent cell viability reagent and plotted using graphing software.Patient-derived organoids can have heterogeneous morphologies. Therefore, it is important to optimize the enzymatic dissociations for every single culture. Downstream analysis of patient-derived models usually involves next-generation sequencing of the DNA and RNA.If possible, validate the molecular characteristics of the models using primary tissue samples. Individualized organoid cultures open up the possibility for personalized medicine approaches for pancreatic cancer patients. Remember that hazardous chemicals such as phenol-chloroform and chemotherapeutic agents must be handled with appropriate PPE and safety equipment.

isolation-characterization-patient-derived-pancreatic-ductal

Research

JoVE Journal

Cancer Research

11.1K vues.  University of California San Diego. Cette méthode est significative parce que les modèles organoïdes patients-dérivés sont un outil puissant et rapide pour étudier le cancer pancréatique car ils reflètent l’hétérogénéité génétique et phénotypique de cette maladie. Le principal avantage de cette technique est que les organoïdes peuvent être isolés avec un taux de réussite élevé à partir d’une quantité limitée de cancer et ils peuvent être étendus rapidement in vitro pour l’analyse en aval. L’é...