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 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 ...

isolation-characterization-patient-derived-pancreatic-ductal

Research

JoVE Journal

Cancer Research

11.1K 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

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

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 ...

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables associated to specific pancreatic pathologies to help preoperative differential diagnosis and patient profiling. Pancreatic juice is a relatively unexplored body fluid, which, due to its close proximity to the tumor site, reflects changes in the surrounding tissue. Here we describe in detail the intraoperative collection procedure. Unfortunately, translating pancreatic juice collection to murine models of PDAC, to perform mechanistic studies, is technically very challenging. Tumor interstitial fluid (TIF) is the extracellular fluid, outside blood and plasma, which bathes tumor and stromal cells. Similarly to pancreatic juice, for its property to collect and concentrate molecules that are found diluted in plasma, TIF can be exploited as an indicator of microenvironmental alterations and as a valuable source of disease-associated biomarkers. Since TIF is not readily accessible, various techniques have been proposed for its isolation. We describe here two simple and technically undemanding methods for its isolation: tissue centrifugation and tissue elution.

Pancreatic juice is a precious source of biomarkers for human pancreatic cancer. We describe here a method for intraoperative collection procedure. To overcome the challenge of adopting this procedure in murine models, we suggest an alternative sample, tumor interstitial fluid, and describe here two protocols for its isolation.

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables ...

Establishment of Zebrafish Patient-Derived Xenografts from Pancreatic Cancer for Chemosensitivity Testing

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms of screening, prevention, and treatment; however, preclinical models that predict the chemosensitivity profile of cancer patients are still lacking. To fill this gap, an in vivo patient-derived xenograft model was developed and validated. The model was based on zebrafish (Danio rerio) embryos at 2 days post fertilization, which were used as recipients of xenograft fragments of tumor tissue taken from a patient's surgical specimen. 
It is also worth noting that bioptic samples were not digested or disaggregated, in order to maintain the tumor microenvironment, which is crucial in terms of analyzing tumor behavior and the response to therapy. The protocol details a method for establishing zebrafish-based patient-derived xenografts (zPDXs) from primary solid tumor surgical resection. After screening by an anatomopathologist, the specimen is dissected using a scalpel blade. Necrotic tissue, vessels, or fatty tissue are removed and then chopped into 0.3 mm x 0.3 mm x 0.3 mm pieces. 
The pieces are then fluorescently labeled and xenotransplanted into the perivitelline space of zebrafish embryos. A large number of embryos can be processed at a low cost, enabling high-throughput in vivo analyses of the chemosensitivity of zPDXs to multiple anticancer drugs. Confocal images are routinely acquired to detect and quantify the apoptotic levels induced by chemotherapy treatment compared to the control group. The xenograft procedure has a significant time advantage, since it can be completed in a single day, providing a reasonable time window to carry out a therapeutic screening for co-clinical trials.

Preclinical models aim to advance the knowledge of cancer biology and predict treatment efficacy. This paper describes the generation of zebrafish-based patient-derived xenografts (zPDXs) with tumor tissue fragments. The zPDXs were treated with chemotherapy, the therapeutic effect of which was assessed in terms of cell apoptosis of the transplanted tissue.

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms ...

Establishment and Characterization of Patient-Derived Xenograft Models of Anaplastic Thyroid Carcinoma and Head and Neck Squamous Cell Carcinoma

Patient-derived xenograft (PDX) models faithfully preserve the histological and genetic characteristics of the primary tumor and maintain its heterogeneity. Pharmacodynamic results based on PDX models are highly correlated with clinical practice. Anaplastic thyroid carcinoma (ATC) is the most malignant subtype of thyroid cancer, with strong invasiveness, poor prognosis, and limited treatment. Although the incidence rate of ATC accounts for only 2%-5% of thyroid cancer, its mortality rate is as high as 15%-50%. Head and neck squamous cell carcinoma (HNSCC) is one of the most common head and neck malignancies, with over 600,000 new cases worldwide each year. Herein, detailed protocols are presented to establish PDX models of ATC and HNSCC. In this work, the key factors influencing the success rate of model construction were analyzed, and the histopathological features were compared between the PDX model and the primary tumor. Furthermore, the clinical relevance of the model was validated by evaluating the in vivo therapeutic efficacy of representative clinically used drugs in the successfully constructed PDX models.

The present protocol establishes and characterizes a patient-derived xenograft (PDX) model of anaplastic thyroid carcinoma (ATC) and head and neck squamous cell carcinoma (HNSCC), as PDX models are rapidly becoming the standard in the field of translational oncology.

Patient-derived xenograft (PDX) models faithfully preserve the histological and genetic characteristics of the primary tumor and maintain its ...

Ovarian Cancer Patient-Derived Organoid Models for Pre-Clinical Drug Testing

Ovarian cancer is a fatal gynecologic cancer and the fifth leading cause of cancer death among women in the United States. Developing new drug treatments is crucial to advancing healthcare and improving patient outcomes. Organoids are in-vitro three-dimensional multicellular miniature organs. Patient-derived organoid (PDO) models of ovarian cancer may be optimal for drug screening because they more accurately recapitulate tissues of interest than two-dimensional cell culture models and are inexpensive compared to patient-derived xenografts. In addition, ovarian cancer PDOs mimic the variable tumor microenvironment and genetic background typically observed in ovarian cancer. Here, a method is described that can be used to test conventional and novel drugs on PDOs derived from ovarian cancer tissue and ascites. A luminescence-based adenosine triphosphate (ATP) assay is used to measure viability, growth rate, and drug sensitivity. Drug screens in PDOs can be completed in 7-10 days, depending on the rate of organoid formation and drug treatments.

We present a protocol that can be used to conduct therapeutic drug testing with patient-derived ovarian cancer organoids.

Ovarian cancer is a fatal gynecologic cancer and the fifth leading cause of cancer death among women in the United States. Developing new drug treatments ...

Automated Kinetic Imaging for Drug Assessment in Patient-Derived Tumor Organoids

Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell lines. PDO models can be generated by culturing patient tumor cells in extracellular basement membrane extracts (BME) and plating them as three-dimensional domes. However, commercially available reagents that have been optimized for phenotypic assays in monolayer cultures often are not compatible with BME. Herein, we describe a method to plate PDO models and assess drug effects using an automated live-cell imaging system. In addition, we apply fluorescent dyes that are compatible with kinetic measurements to quantify cell health and apoptosis simultaneously. Image capture can be customized to occur at regular time intervals over several days. Users can analyze drug effects in individual Z-plane images or a Z Projection of serial images from multiple focal planes. Using masking, specific parameters of interest are calculated, such as PDO number, area, and fluorescence intensity. We provide proof-of-concept data demonstrating the effect of cytotoxic agents on cell health, apoptosis, and viability. This automated kinetic imaging platform can be expanded to other phenotypic readouts to understand diverse therapeutic effects in PDO models of cancer.

Author Spotlight: Developing Multiplexed Kinetic Assays for Organoid-Based Drug Response Analysis

Patient-derived tumor organoids are a sophisticated model system for basic and translational research. This methods article details the use of multiplexed fluorescent live-cell imaging for simultaneous kinetic assessment of different organoid phenotypes.

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell ...

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 ...