Name: isolation and characterization of patient-derived pancreatic ductal adenocarcinoma organoid models
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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 expansion. Both conditioned mediums described below are prepared from commercially available cell lines (see Table of Materials). For complete protocols and materials, please refer to the manufacturers&#39; websites.
	<ol>
		<li>Produce R-spondin1 conditioned media according to the manufacturer&#39;s protocol.
		<ol>
			<li>First, expand the 293T cells expressing R-spondin1 using appropriate antibiotic (Zeocin) selection methods in the presence of abundant serum (10%). When producing conditioned media, withdraw and wash away the antibiotic selection and serum such that no antibiotic and serum are present in the final conditioned media. Importantly, the conditioned media must be filter sterilized after collection (0.2 &#181;m) to prevent cross-contamination.</li>
			<li>Store aliquoted conditioned media at 4 &#176;C for short term use (within 6 months) or at -20 &#176;C for long term storage (more than 6 months). Freeze-thaw cycles should be avoided.</li>
		</ol>
		</li>
		<li>Produce L-Wnt-3A conditioned media according to the manufacturer&#39;s protocol.
		<ol>
			<li>First, expand the L-M(TK-) cells expressing L-Wnt-3A using appropriate antibiotic (G-418) selection methods in the presence of abundant serum (10%). When producing conditioned media, withdraw and wash away the antibiotic selection such that no antibiotic is present in the final conditioned media; however, maintain the serum throughout the culturing and conditioning. Importantly the collected conditioned media must be filter sterilized (0.2 &#181;m) to prevent cross contamination.</li>
			<li>Store the aliquoted conditioned media at 4 &#176;C for short term use, or -20 &#176;C for long term storage. Freeze-thaw cycles should be avoided.</li>
		</ol>
		</li>
		<li>For quality control, perform a TOPFLASH assay to test the Wnt activity of R-spondin1 and the L-Wnt-3A conditioned media alone and in combination according to a previously published protocol14.</li>
	</ol>
	</li>
	<li>Basal media preparation: Use basal media for preparation of complete organoid media as well as washing steps for organoid work. Supplement advanced DMEM/F-12 with Glutamine at a final concentration of 1x, HEPES (10 mM), and Penicillin/Streptomycin (100 U/mL). Store basal media at 4 &#176;C.</li>
	<li>Organoid Complete media preparation: Supplement the basal media with R-spondin1 conditioned media at a final concentration of 10% v/v, L-Wnt-3A conditioned media at a final concentration of 50% v/v, Human EGF (50 ng/mL), Human FGF (100 ng/mL), Human Gastrin I (10 nM), Mouse Noggin (100 ng/mL), A83-01 (500 nM), B27 supplement (1x), Nicotinamide (10 mM), N-acetylcysteine (1.25 mM), and Primocin (100 &#181;g/mL).
	<ol>
		<li>For primary organoid isolations, thawed organoid cultures, and cultures starting with single cells, include Rho kinase inhibitor Y-27632 at a final concentration of 10.5 &#181;M.</li>
		<li>Prepare the human organoid complete media on ice and store at 4 &#176;C for use within one month. For this reason, we recommend fresh weekly preparation of media.</li>
	</ol>
	</li>
</ol>
2. Isolation of PDAC Organoids
NOTE: Thaw the basement membrane extract (BME) solution (growth factor reduced; see Table of Materials) on ice in a 4 &#176;C environment (fridge or cold room) for at least 12 h prior to use. Incubate the tissue culture plates for organoid culture in a 37 &#176;C incubator for at least 12 h prior to use.
<ol>
	<li>Collect and transport a surgically removed tumor specimen for research in a buffered storage solution to maintain viability of the tissue. Use basal media or a commercially available tissue storage solution.</li>
	<li>Proceed to isolate organoids as soon as possible after receiving the specimen. Tissue can be stored in the storage solution up to 24 h post-surgery and still yield organoids.</li>
	<li>Once in the laboratory, transfer tumor to a 10 cm tissue culture dish and remove storage solution.</li>
	<li>While handling the tissue with metallic forceps, mince the tumor with a #10 scalpel into small fragments of 1 mm3 or smaller. Larger fragments will take longer to digest; therefore, aim to generate homogenous tissue fragment sizes. If tissue is already disaggregated or in smaller than 1 mm3 fragments, skip this step.</li>
	<li>Transfer the tissue fragments to a 15 mL conical tube containing 10 mL of basal media. Mix by inverting the tube several times. Centrifuge the tube at 200 x g for 5 min. After spin, remove the basal media carefully to avoid losing tissue fragments.</li>
	<li>To the tube containing the tissue fragments add 9 mL of basal media and 1 mL of 10x Gentle Collagenase/Hyaluronidase solution. To avoid clumping of cells during digestion, supplement this solution with 20 &#181;L of 10 mg/mL DNAse I solution. Mix by inverting the tube several times.</li>
	<li>Incubate the enzymatic digestion at 37 &#176;C on a nutator or rotator. For adequate mixing during digestion, the tissue fragments must constantly be moving through the solution. 
	NOTE: Timing for this enzymatic dissociation of the tumor must be determined empirically for every specimen. A successful digestion of the tumor is characterized by the decrease in size of the tissue fragments until they are not clearly discernable to the naked eye. Concomitantly, the opacity of the solution will increase as microscopic tissue fragments are released.</li>
	<li>After 30 min, remove the conical tube from the 37 &#176;C incubator and observe. If the tissue fragments are still clearly visible, continue the dissociation at 37 &#176;C with constant mixing. Repeat this observation every 30 min until most or all tissue fragments have been dissociated. Depending on the stringency of mixing as well as the size and amount of tumor fragments, this step can take as little as 30 min for small specimens or as long as 12 h for larger tumor samples.</li>
	<li>Once the enzymatic dissociation is complete, centrifuge the tube at 200 x g for 5 min. After spin, a cell pellet should be visible and the supernatant should be clear, indicating that all cells have been spun out of solution. If that is not the case, repeat the spin.</li>
	<li>Carefully remove the supernatant to avoid losing the cell pellet. Immediately wash the cells with 10 mL basal media by gently mixing the tubes with inversion. Centrifuge the tube at 200 x g for 5 min. Carefully remove the basal media to avoid losing cells and repeat this washing step one more time for a total of two washes.</li>
	<li>After the second wash, remove all of the basal media carefully and place the tube on ice for 5 min.</li>
	<li>At this time, place an aliquot of organoid complete media in a 37 &#176;C water bath to pre-warm.</li>
	<li>Mix the cell pellet with ice cold BME while maintaining the tube on ice. Seeding density should be high for organoid isolation. For a small pellet (~50 &#181;L volume) use 200 &#181;L of BME, while for a large pellet (~200 &#181;L volume) use 800 &#181;L of BME. Mix the cells and BME on ice using a p200 pipette until the solution appears homogenous while avoiding creating bubbles in the solution.</li>
	<li>Spot a 100 &#181;L dome in the center of a well of a pre-warmed 12 well plate using a p200 pipette. Repeat this until all of the BME solution have been dispensed. Carefully transfer the plate to the 37 &#176;C incubator to allow the BME gel to solidify.</li>
	<li>After 10 min, retrieve the plate and add 1 mL of pre-warmed organoid complete media per well. Dispense the media on the side of the well to avoid disruption of the BME dome.</li>
	<li>Observe the cell fragments using an inverted tissue culture microscope using a 4x lens in brightfield. 
	NOTE: Single cells and microscopic tissue fragments should be clearly visible. A successful isolation is characterized by the appearance of more than 10 organoids after 24&#8211;48 h; the culture should be confluent within one week. As tumor specimens are heterogenous, some organoid isolations are more challenging as only a few organoids grow per well, as shown in Figure 1. In this case, allow the culture to continue for up to two weeks to maximize the number of cells available for passaging. To account for media consumption and evaporation during culture, top up the cultures with 200 &#181;L of pre-warmed organoid complete media every 5 days.</li>
</ol>
3. Passaging of PDAC Organoids
<ol>
	<li>Retrieve the plate from tissue culture incubator. With a sterile cell lifter, gently lift the BME dome such that it is floating in the complete media. Avoid scraping the bottom of the well so as to not remove any monolayer cells attached to the plastic, as these are typically fibroblasts.</li>
	<li>With a p1000 pipette, carefully transfer the BME dome and media to a 15 mL conical tube. Repeat this step for every organoid-containing well.</li>
	<li>Gently wash each well that was harvested with 1 mL of cold basal media, and transfer to the 15 mL tube containing the organoids.</li>
	<li>Thoroughly mix the solution and organoids with a p1000 pipette and spin down at 200 x g for 5 min. After spinning, a BME layer containing organoids in suspension and an organoid pellet will appear at the bottom of the tube. Carefully remove the supernatant, avoiding loss of the BME/organoid layer. Place the tube on ice.</li>
	<li>Add 10 mL of ice-cold cell recovery solution (CRS) to the tube and thoroughly mix by inversion. This will depolymerize the protein matrices of the gelled BME at 4 &#176;C. Incubate on ice for 30 min while mixing every 3 min by inversion. If available, place the tube on a rotating mixer in a cold room or 4 &#176;C fridge for 30 min.</li>
	<li>After the 30 min incubation, spin down at 200 x g for 5 min. 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 min at 4 &#176;C with mixing and repeat spin.</li>
	<li>Once the BME has been depolymerized, remove the CRS leaving behind the organoid pellet. Wash the organoids with 10 mL basal media by mixing with inversion. Spin down at 200 x g for 5 min and remove the supernatant. Place the tube with the organoid pellet on ice for at least 5 min.</li>
	<li>Optionally, if a single cell preparation is desired, incubate the organoids with 3 mL trypsin supplemented with 30 &#181;L of 10 mg/mL DNAse I solution to avoid clumping of cells during digestion.
	<ol>
		<li>Incubate the enzymatic reaction at 37 &#176;C with gentle inversion mixing every 2 min for up to 10 min. Monitor and confirm successful single cell dissociation under brightfield microscope.</li>
		<li>To stop the enzymatic reaction, add 10 mL of ice cold basal media and spin down at 200 x g for 5 min.</li>
		<li>Wash cells with 10 mL basal media by mixing with gentle inversion. Spin down at 200 x g for 5 min and remove the supernatant and repeat the wash one more time. Place the tube with the cell pellet on ice for at least 5 min. 
		NOTE: We recommend performing this step every 3-5 passages during regular maintenance of the organoids to generate homogeneous culture with a large number of organoids.</li>
	</ol>
	</li>
	<li>Add ice cold BME to the cell pellet and mix gently on ice until solution is homogenous using a p200 pipette. While avoiding generating bubbles in the mixture, pipette up and down at least 5&#8211;10x, placing the tip of the pipette close to the bottom of the tube to help mechanically break up the organoids. As a guide, use 4&#8211;6x the volume of the BME/cell pellet such that the splitting ratio is no more than 1:2 from one passage to the other.</li>
	<li>Spot a 100 &#181;L dome in the center of a well of a pre-warmed 12 well plate using a p200 pipette; repeat until all of the BME solution has been dispensed. Carefully transfer the plate to the 37 &#176;C incubator to allow the BME gel to solidify. After 10 min, retrieve the plate and add 1 mL of pre-warmed organoid complete media per well. Dispense the media on the side of the well to avoid disruption of the BME dome.</li>
	<li>Organoids should reform and start growing within 24 h. Organoid cultures are typically passaged ever 7-10 days depending on the culture density and cell proliferation. If necessary, top up the cultures with 200 &#181;L of pre-warmed organoid complete media every 5 days to compensate for growth factor depletion and evaporation.</li>
</ol>
4. Freezing and Thawing of PDAC Organoids
<ol>
	<li>To freeze down the organoids, proceed to harvest the organoids and depolymerize the BME as described in steps 3.1&#8211;3.7.</li>
	<li>To the cell pellet, add 1 mL of freezing media, mix gently, and transfer the mixture to a pre-labeled cryovial.</li>
	<li>Place the cryovial in a freezing container to maintain a safe constant temperature decrease and place in a -80 &#176;C freezer. After 24 to 48 h, transfer the frozen vials to liquid nitrogen storage. Organoids can be cryopreserved for months to years using this method.</li>
	<li>To thaw the organoids, retrieve the frozen cryovial from liquid nitrogen storage. Transport the frozen vial on dry ice to the tissue culture room.</li>
	<li>Place the frozen cryovial in the 37 &#176;C water bath to rapidly thaw the organoids and transfer the contents of the vial to a 15 mL conical tube containing 9 mL of basal media warmed to room temperature.</li>
	<li>Spin down at 200 x g for 5 min and remove the supernatant. Place the tube with the organoid pellet on ice for at least 5 min.</li>
	<li>Proceed to resuspend in BME and plate the organoids as described in steps 3.9&#8211;3.11. 
	NOTE: Organoid cultures recover slowly after a freeze/thaw cycle, therefore cultures may be grown for up to two weeks before passaging.</li>
</ol>
5. Characterization of PDAC Organoids
NOTE: The characterization of the organoids should be performed on an established culture after several passages to diminish the risk of contamination from non-epithelial cell types such as fibroblasts and immune cells.
<ol>
	<li>Nucleic acid extraction from PDAC organoids
	<ol>
		<li>Plate organoids on a 12 well plate dedicated to nucleic acid extraction. Plate no more than 100 &#181;L of BME per well. For adequate DNA/RNA yield, plate at least 2&#8211;4 wells per culture. Grow organoids for up to 5 days until cultures are close to confluent but devoid of the excessive cell debris that accumulates in longer term cultures.</li>
		<li>Retrieve the plate from the 37 &#176;C incubator and completely remove all trace of organoid complete media, leaving only the BME dome containing the organoids on the plate.</li>
		<li>Add 900 &#181;L of acid phenol reagent (see Table of Materials) to each well and mix thoroughly using a p1000 pipette until solution becomes homogeneous. The BME will be completely dissolved in the acid phenol reagent and organoids will lyse after a short 5 min incubation. 
		CAUTION: Acid phenol is a hazardous chemical that can cause chemical burns. Handle with care using appropriate protections and technique.</li>
		<li>Transfer the homogenous solution to a 2 mL tube and add 200 &#181;L of chloroform. Incubate 5 min and centrifuge at 12,000 x g for 15 min at 4 &#176;C.</li>
		<li>Proceed to the RNA and DNA extraction according to manufacturer&#39;s protocol. RNA can be extracted from the aqueous phase while DNA can be extracted from the interphase and organic phase. Once purified and quantified, RNA and DNA isolated from different wells of the same culture can be pooled to increase yield. 
		NOTE: (1) While we strongly recommend using this nucleic acid extraction method for RNA, there are many good methods for isolating DNA from cultured cells. (2) To ascertain the presence of tumor cells within the organoid culture, we recommend sequencing the DNA using your preferred next generation sequencing method to identify mutations within PDAC hallmark genes (KRAS, TP53, SMAD4, CDKN2A).</li>
	</ol>
	</li>
	<li>Pharmacotyping of PDAC organoids
	<ol>
		<li>Isolate single cells from organoids as described above in steps 3.1&#8211;3.8. To maximize cell number, harvest at least 10 confluent wells of a 12 well plate</li>
		<li>Resuspend single cells in 1 mL human organoid complete media. The presence of small cell clumps (~2&#8211;10 cells) is acceptable, however larger cell clumps will negatively affect the downstream analysis.</li>
		<li>Count cells using an automated cell counter and record cell viability. Calculate the total number of cells and viable cells present in the 1 mL suspension.</li>
		<li>For therapeutic testing, plate 1,000 viable cells per well of a 384 well plate. For a 384 well plate we estimate we will use 400,000 viable cells (calculated for 400 wells).</li>
		<li>Prepare cells for plating by mixing on ice 800 &#181;L of BME with 7.2 mL of organoid complete media containing the cells. Keep mixture on ice and plate 20 &#181;L per well of a 384 well plate. Use a 12-channel pipette and reservoir kept on ice to efficiently plate the cells. To avoid excessive evaporation from the plate, the outer wells can be used as a reservoir (60 &#181;L PBS or water per well), but this will lead to a loss of 76 experimental wells.</li>
		<li>Spin the plate down at 100 x g for 1 min in a swing bucket. This allows the small 20 &#181;L volume and organoids to settle at the bottom of the plate.</li>
		<li>Place plate in the 37 &#176;C tissue culture incubator to allow organoids to form. After 24 h,check for presence of organoids using a brightfield microscope.</li>
		<li>Proceed to dosing therapeutic compounds dissolved in DMSO onto the plate using a drug printer or equivalent instrument. Test each drug dose in at least triplicate wells. To perform an effective dose-response analysis, determine dose range for each drug empirically, starting with a low, ineffective, dose and ending with a high dose where maximum effect is observed. Examples of dose ranges for chemotherapeutic compounds have been recently published9.</li>
		<li>Expose the cells to the therapeutic compounds for 3&#8211;5 days.</li>
		<li>Optionally, at the end of the assay, image the plate to evaluate the therapeutic effect on organoid size, number, and morphology.</li>
		<li>Assess viability using 20 &#181;L per well of luminescence cell viability reagent (see Table of Materials) according to manufacturer&#39;s protocol. A luminometer will be necessary for data acquisition and a graphing software for data analysis.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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><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 &amp;deg;C CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td></td><td></td><td></td></tr><tr><td>37 &amp;deg;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 &amp;deg;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 &amp;micro;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 ...

isolation-characterization-patient-derived-pancreatic-ductal

Research

JoVE Journal

Cancer Research

11.2K Views.  University of California San Diego. 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.

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

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Creating Matched In vivo/In vitro Patient-Derived Model Pairs of PDX and PDX-Derived Organoids for Cancer Pharmacology Research

Patient-derived tumor xenografts (PDXs) are considered the most predictive preclinical models, largely believed to be driven by cancer stem cells (CSC) for conventional cancer drug evaluation. A large library of PDXs is reflective of the diversity of patient populations and thus enables population based preclinical trials (&#8220;Phase II-like mouse clinical trials&#8221;); however, PDX have practical limitations of low throughput, high costs and long duration. Tumor organoids, also being patient-derived CSC-driven models, can be considered as the in vitro equivalent of PDX, overcoming certain PDX limitations for dealing with large libraries of organoids or compounds. This study describes a method to create PDX-derived organoids (PDXO), thus resulting in paired models for in vitro and in vivo pharmacology research. Subcutaneously-transplanted PDX-CR2110 tumors were collected from tumor-bearing mice when the tumors reached 200-800 mm3, per an approved autopsy procedure, followed by removal of the adjacent non-tumor tissues and dissociation into small tumor fragments. The small tumor fragments were washed and passed through a 100 &#181;m cell strainer to remove the debris. Cell clusters were collected and suspended in basement membrane extract (BME) solution and plated in a 6-well plate as a solid droplet with surrounding liquid media for growth in a CO2 incubator. Organoid growth was monitored twice weekly under light microscopy and recorded by photography, followed by liquid medium change 2 or 3 times a week. The grown organoids were further passaged (7 days later) at a 1:2 ratio by disrupting the BME embedded organoids using mechanical shearing, aided by addition of trypsin and the addition of 10 &#181;M Y-27632. Organoids were cryopreserved in cryo-tubes for long-term storage, after release from BME by centrifugation, and also sampled (e.g., DNA, RNA and FFPE block) for further characterization.

A method is described to create organoids using patient-derived xenografts (PDX) for in vitro screening, resulting in matched pairs of in vivo/in vitro models. PDX tumors were harvested/processed into small pieces mechanically or enzymatically, followed by the Clevers&#8217; method to grow tumor organoids that were passaged, cryopreserved and characterized against the original PDX.

Patient-derived tumor xenografts (PDXs) are considered the most predictive preclinical models, largely believed to be driven by cancer stem cells (CSC) ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Developmental Biology

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex vivo drug screenings. However, current adenosine triphosphate (ATP)-based drug screening assays do not capture the complexity of a drug response (cytostatic or cytotoxic) and intratumor heterogeneity that has been shown to be retained in PDTOs due to a bulk readout. Live-cell imaging is a powerful tool to overcome this issue and visualize drug responses more in-depth. However, image analysis software is often not adapted to the three-dimensionality of PDTOs, requires fluorescent viability dyes, or is not compatible with a 384-well microplate format. This paper describes a semi-automated methodology to seed, treat, and image PDTOs in a high-throughput, 384-well format using conventional, widefield, live-cell imaging systems. In addition, we developed viability marker-free image analysis software to quantify growth rate-based drug response metrics that improve reproducibility and correct growth rate variations between different PDTO lines. Using the normalized drug response metric, which scores drug response based on the growth rate normalized to a positive and negative control condition, and a fluorescent cell death dye, cytotoxic and cytostatic drug responses can be easily distinguished, profoundly improving the classification of responders and non-responders. In addition, drug-response heterogeneity can by quantified from single-organoid drug response analysis to identify potential, resistant clones. Ultimately, this method aims to improve the prediction of clinical therapy response by capturing a multiparametric drug response signature, which includes kinetic growth arrest and cell death quantification.

This protocol describes a semi-automated method for medium- to high-throughput organoid drug screenings and microscope-agnostic, automated image analysis software to quantify and visualize multiparametric, single-organoid drug responses to capture intratumor heterogeneity.

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex ...

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 ...

Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts from Fresh Tissue

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

Author Spotlight: Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue  

Tumor organoids have revolutionized cancer research and the approach to personalized medicine. They represent a clinically relevant tumor model that allows researchers to stay one step ahead of the tumor in the clinic. This protocol establishes tumor organoids from fresh pancreatic tumor tissue samples and patient-derived xenografts of pancreatic adenocarcinoma origin.

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. ...

Reprogramming PDAC and Pancreatic Cells into Induced Pluripotent Stem Cells

Reprogramming Pancreatic Ductal Adenocarcinoma to Pluripotency

The generation of induced pluripotent stem cells (iPSCs) using transcription factors has been achieved from almost any differentiated cell type and has proved highly valuable for research and clinical applications. Interestingly, iPSC reprogramming of cancer cells, such as pancreatic ductal adenocarcinoma (PDAC), has been shown to revert the invasive PDAC phenotype and override the cancer epigenome. The differentiation of PDAC-derived iPSCs can recapitulate PDAC progression from its early pancreatic intraepithelial neoplasia (PanIN) precursor, revealing the molecular and cellular changes that occur early during PDAC progression. Therefore, PDAC-derived iPSCs can be used to model the earliest stages of PDAC for the discovery of early-detection diagnostic markers. This is particularly important for PDAC patients, who are typically diagnosed at the late metastatic stages due to a lack of reliable biomarkers for the earlier PanIN stages. However, reprogramming cancer cell lines, including PDAC, into pluripotency remains challenging, labor-intensive, and highly variable between different lines. Here, we describe a more consistent protocol for generating iPSCs from various human PDAC cell lines using bicistronic lentiviral vectors. The resulting iPSC lines are stable, showing no dependence on the exogenous expression of reprogramming factors or inducible drugs. Overall, this protocol facilitates the generation of a wide range of PDAC-derived iPSCs, which is essential for discovering early biomarkers that are more specific and representative of PDAC cases.

Author Spotlight: Reprogramming Cancer Cells to iPSCs to Study Disease Progression and Treatment Targets

The present protocol describes the reprogramming of Pancreatic Ductal Adenocarcinoma (PDAC) and normal pancreatic ductal epithelial cells into induced pluripotent stem cells (iPSCs). We provide an optimized and detailed, step-by-step procedure, from preparing lentivirus to establishing stable iPSC lines.

The generation of induced pluripotent stem cells (iPSCs) using transcription factors has been achieved from almost any differentiated cell type and has ...

Patient-Derived Melanoma Organoids: A Frontier in Cancer Immunotherapy

Unveiling Therapeutic Opportunities with Melanoma Patient-derived Organoid Models

With the development of immunotherapy, there is an ongoing need to develop models that can recapitulate the tumor microenvironment of native tumors. While traditional two- and three-dimensional models can offer insights into cancer development and progression, these lack crucial aspects that hinder a faithful mimic of native tumors. An alternative model that has gained a lot of attention is the patient-derived organoid. The development of these organoids recapitulates the complex intercellular communication, tumor microenvironment, and histoarchitecture of tumors. This paper describes the protocol for establishing melanoma patient-derived organoid (MPDO) models. To validate these models, we assessed the immune cell composition, including the expression levels of T-cell activation markers, to confirm the cellular heterogeneity of the organoids. Additionally, to describe the potential utility of MPDOs in cellular therapies, we evaluated the cytotoxic capabilities of treating the organoids with &#947;&#948; T-cells. In conclusion, the MPDO models offer promising avenues for understanding tumor complexity, validating therapeutic strategies, and potentially advancing personalized treatment.

Author Spotlight: Recreating Melanoma Complexity with Patient-Derived Organoids for Immunotherapy Evaluation

Here, we present a protocol to generate melanoma patient-derived organoids by culturing disassociated cell suspensions from fresh melanoma tissues. These organoids faithfully recapitulate patient-specific tumors in vitro, offering an innovative approach to exploring tumor immunosuppressive mechanisms, drug screening, drug resistance mechanisms, and cancer surveillance approaches.

With the development of immunotherapy, there is an ongoing need to develop models that can recapitulate the tumor microenvironment of native tumors. While ...

A Preclinical Human-Derived Autologous Gastric Cancer Organoid/Immune Cell Co-Culture Model to Predict the Efficacy of Targeted Therapies

Tumors expressing programmed cell death-ligand 1 (PD-L1) interact with programmed cell death protein 1 (PD-1) on CD8+ cytotoxic T lymphocytes (CTLs) to evade immune surveillance leading to the inhibition of CTL proliferation, survival, and effector function, and subsequently cancer persistence. Approximately 40% of gastric cancers express PD-L1, yet the response rate to immunotherapy is only 30%. We present the use of human-derived autologous gastric cancer organoid/immune cell co-culture as a preclinical model that may predict the efficacy of targeted therapies to improve the outcome of cancer patients. Although cancer organoid co-cultures with immune cells have been reported, this co-culture approach uses tumor antigen to pulse the antigen-presenting dendritic cells. Dendritic cells (DCs) are then cultured with the patient's CD8+ T cells to expand the cytolytic activity and proliferation of these T lymphocytes before co-culture. In addition, the differentiation and immunosuppressive function of myeloid-derived suppressor cells (MDSCs) in culture are investigated within this co-culture system. This organoid approach may be of broad interest and appropriate to predict the efficacy of therapy and patient outcome in other cancers, including pancreatic cancer.

The goals of the protocol are to use this approach to 1) understand the role of the immunosuppressive gastric tumor microenvironment and 2) predict the efficacy of patient response, thus increasing the survival rate of patients.

Tumors expressing programmed cell death-ligand 1 (PD-L1) interact with programmed cell death protein 1 (PD-1) on CD8+ cytotoxic T lymphocytes (CTLs) to ...

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

Summary

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Chapters in this video

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

Creating Matched In vivo/In vitro Patient-Derived Model Pairs of PDX and PDX-Derived Organoids for Cancer Pharmacology Research

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Establishment and Culture of Patient-Derived Breast Organoids

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

Reprogramming Pancreatic Ductal Adenocarcinoma to Pluripotency

Unveiling Therapeutic Opportunities with Melanoma Patient-derived Organoid Models

A Preclinical Human-Derived Autologous Gastric Cancer Organoid/Immune Cell Co-Culture Model to Predict the Efficacy of Targeted Therapies