We are grateful for the guidance of Ron Bose, MD, PhD, and the assistance of Barbara Blachut, MD, in establishing this protocol. We would also like to acknowledge Washington University&#39;s School of Medicine in St. Louis&#39;s Department of Obstetrics and Gynecology and Division of Gynecologic Oncology, Washington University&#39;s Dean&#39;s Scholar Program, and the Reproductive Scientist Development Program for their support of this project.

All human tissue specimens collected for research were obtained according to the Institutional Review Board (IRB)-approved protocol. The protocols outlined below were performed in a sterile human tissue culture environment. Informed written consent was obtained from human subjects. Eligible patients had to have a diagnosis or presumed diagnosis of ovarian cancer, be willing and able to sign informed consent, and be at least 18 years of age. Tumor tissue (malignant primary tumor or metastatic sites), ascites, and pleural fluid were obtained from consenting patients at the time of their procedure. These specimens were immediately transported to the laboratory and processed for organoid generation using the methods outlined below.
1. Media preparation
<ol>
	<li>Complete organoid media preparation
	<ol>
		<li>Prepare R-spondin 1/Noggin conditioned medium following a previously published report7. 
		NOTE: R-spondin-1/Noggin conditioned medium is a more affordable alternative to commercially available recombinant proteins. HEK293T cells stably secreting R-spondin-1 and Noggin via lentivirus-mediated transduction were a generous gift from Ron Bose, Washington University School of Medicine in St. Louis, and Anil Rustgi, New York-Presbyterian/Columbia University Irving Medical Center8,9,10. Commercial conditioned medium could be used as a substitute (see Table of Materials).</li>
	</ol>
	</li>
	<li>To make the complete organoid medium, combine 10% R-spondin 1/Noggin conditioned medium, 50 ng/mL EGF, 10 ng/mL FGF-10, 10 ng/mL FGF2, 1x B27, 10 mmol/L nicotinamide, 1.25 mmol/L N-acetylcysteine, 1 &#181;mol/L prostaglandin E2, 10 &#181;mol/L SB202190, 500 nmol/L A83-01, and 10 &#181;M ROCK inhibitor (see Table of Materials). 
	&#8203;NOTE: The medium may be stored at 4 &#176;C for up to 3 months. This medium was adapted from Hill et al.11. The concentrations and ingredients of the medium are the same, with the addition of a ROCK inhibitor.</li>
	<li>Prepare organoid base medium by combining 500 mL of an advanced formulation of DMEM/F12 with 1% penicillin-streptomycin, 1x dipeptide, L-alanyl-L-glutamine, and 1x HEPES (10 mM) (see Table of Materials).</li>
</ol>
2. Harvesting organoids from ascites and pleural fluid
NOTE: Ascites and pleural fluid must be processed as soon as possible for the best yield of organoids. Thaw previously aliquoted BME, DNase I, and DNase I reaction buffer (see Table of Materials) by placing it on ice until the contents are liquefied.
<ol>
	<li>Obtain ascites and pleural fluid from consenting patients at the time of standard care surgeries or procedures, and transport at room temperature in a travel container to the laboratory. 
	NOTE: All ascites or pleural fluid processing should be performed in a sterile environment.</li>
	<li>Transfer 50 mL of ascites or pleural fluid to 50 mL conical tubes (the number of tubes depends on the volume of ascites obtained). Centrifuge at 1,650 x g for 5 min at 4 &#176;C. After centrifugation, use a glass Pasteur pipette to carefully aspirate the supernatant.</li>
	<li>Continue by adding 50 mL of ascites or pleural fluid to the previously centrifuged pellet, and centrifuge again at 1,650 x g for 5 min at 4 &#176;C. Carefully aspirate the supernatant using a glass Pasteur pipette. Repeat this step until all the ascites or pleural fluid have been processed.</li>
	<li>Prepare a 100 &#181;g/mL DNase I solution by combining 1,000 &#181;L of nuclease-free water, 100 &#181;L of DNase I reaction buffer, and 10 &#181;L of DNase I. 
	NOTE: The DNase I treatment applied is sufficient to make a single-cell suspension regardless of the presence of cell aggregates in some patient samples12.</li>
	<li>Resuspend each cell pellet in 1 mL of 100 &#181;g/mL DNase I solution. Gently add the DNase I solution dropwise, and allow the tube to incubate for 15 min at room temperature. 
	NOTE: Add a minimum of 1 mL of DNase I solution. If 1 mL is not enough to disturb the pellet, add an additional 1 mL.</li>
	<li>After incubation, add 25 mL of organoid base medium (step 1.3) to the cells, and gently invert to mix. Then, centrifuge at 1,650 x g for 5 min at 4 &#176;C. After centrifugation, carefully aspirate the supernatant using a glass Pasteur pipette.</li>
	<li>Resuspend the newly formed cell pellets in pre-warmed 5 mL of 1x red blood cell (RBC) lysis buffer (see Table of Materials). Set the vortex to 458 x g, and vortex the solutions in each conical tube. Once the solutions are homogenous, use a serological pipet to combine the contents of all the conical tubes into a single 50 mL conical tube.</li>
	<li>Incubate the conical tube containing the vortexed solution at room temperature for 5 min. Once incubation is complete, centrifuge at 1,650 x g for 5 min at 4 &#176;C. 
	NOTE: Examine the pellet. A pink/red pellet indicates the presence of RBCs, which would require the RBC lysis buffer step to be repeated until the pellet is no longer red.</li>
	<li>After centrifugation, carefully aspirate the supernatant using a glass Pasteur pipette. Then, wash the pellet with 10 mL of PBS, vortex the solution at 458 x g, and centrifuge at 1,650 x g for 5 min at 4 &#176;C.</li>
	<li>If a large cell pellet is formed, aspirate the PBS using a glass Pasteur pipette, and add 1 mL of organoid base medium (step 1.3) on top of the pellet. Vortex the solution at 458 x g, and transfer 300-400 &#181;L to a microcentrifuge tube. Centrifuge the microcentrifuge tube at 1,650 x g for 5 min at 4 &#176;C. 
	NOTE: The portion of the cell pellet not placed in the microcentrifuge tube can be frozen down for future use (500 &#181;L of cells to 1 mL of 10% DMSO in FBS). The frozen cell pellet can be stored for weeks at &#8722;80 &#176;C and for years if placed in liquid nitrogen13.</li>
	<li>Carefully aspirate the organoid base medium (step 1.3) using a glass Pasteur pipette, and resuspend in BME (see Table of Materials) using cold tips. 
	NOTE: The amount of BME is based on the size of the pellet. It is recommended to use 25% organoid base medium (step 1.3) with resuspended cells to 75% BME.</li>
	<li>Plate 40 &#181;L aliquots of the resuspended cell solution onto a 6-well plate. Plate up to five aliquots per well (Figure 1).</li>
	<li>Immediately place the plate in a 37 &#176;C incubator for 20 min to allow the BME to solidify. After incubation, gently add 2 mL of complete organoid medium (step 1.2) into each well.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64878/64878fig01.jpg" /> 
Figure 1: Plating of patient-derived ovarian cancer organoids. Representative image of the organoid plating. Aliquots of the organoid mixture are carefully plated, ensuring no bubbles are formed. <a href="https://www.jove.com/files/ftp_upload/64878/64878fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Harvesting organoids from tissue
NOTE: Tissue must be processed as soon as possible for the best yield of organoids.
<ol>
	<li>Collect and transport the tumor on ice in a travel container containing PBS.</li>
	<li>Place the sample on a 10 cm tissue culture dish (Figure 2). Using a disposable scalpel, mince the tissue. Next, using the dull end of a disposable syringe, crush the tissue until a homogenous mixture has been created.</li>
	<li>Using forceps, place the homogenous tissue mixture into a dissociation tube. For every 1-2 mL of homogenized tissue, add 7-8 mL of 1 mg/mL type II collagenase solution (see Table of Materials) in organoid base medium (step 1.3) and 1 mL of DNase I solution. Vortex the solution at 458 x g. 
	NOTE: The amount of tissue will determine the amount of collagenase solution and the number of tubes needed.</li>
	<li>Use the dissociation machine (see Table of Materials) to liquefy the tissue while it is in the collagenase solution. Run the program 37C_h_TDK3 (1 h) until it is a single-cell suspension. 
	NOTE: The program will need to be rerun if the resulting mixture is not homogenous (i.e., if tissue pieces are still present in the mixture). If the tissue is digested but the solution is viscous, dilute using organoid base medium (step 1.3).</li>
	<li>Transfer the homogenized mixture to a new 50 mL conical tube, and add 20-40 mL of organoid base medium (step 1.3). Then, filter the solution through a 100 &#181;m cell strainer into a newly labeled 50 mL conical tube.</li>
	<li>Centrifuge the filtered mixture at 1,650 x g for 5 min at 4 &#176;C. Then, carefully aspirate the supernatant using a glass Pasteur pipette.</li>
	<li>Prepare a 100 &#181;g/mL DNase I solution by combining 1,000 &#181;L of nuclease-free water, 100 &#181;L of DNase I reaction buffer, and 10 &#181;L of DNase I. 
	NOTE: The DNase I treatment applied is sufficient to make a single-cell suspension regardless of the presence of cell aggregates in some patient samples12.</li>
	<li>Resuspend the cells in 1 mL of 100 ug/mL DNase I solution. Gently add the DNase I solution dropwise, and allow the tube to incubate for 15 min at room temperature.</li>
	<li>After incubation, add 25 mL of organoid base medium (step 1.3) to the cells, and gently invert to mix. Then, centrifuge at 1,650 x g for 5 min at 4 &#176;C. After centrifugation, carefully aspirate the supernatant using a glass Pasteur pipette.</li>
	<li>Resuspend the newly formed cell pellet in 5 mL of pre-warmed 1x RBC lysis buffer. Vortex the solutions in each conical tube at 458 x g.</li>
	<li>Incubate the conical tube containing the cell pellet resuspended in the RBC lysis buffer for 5 min. Once the incubation is complete, centrifuge at 1,650 x g for 5 min at 4 &#176;C. 
	NOTE: Examine the pellet. A pink/red pellet indicates the presence of RBCs, which would require the RBC lysis buffer step to be repeated until the pellet appears white.</li>
	<li>After centrifugation, carefully aspirate the supernatant using a glass Pasteur pipette. Then, wash the pellet with 10 mL of PBS, vortex the solution at 458 x g, and centrifuge at 1,650 x g for 5 min at 4 &#176;C.</li>
	<li>If a large cell pellet is formed, aspirate the PBS using a glass Pasteur pipette, and add 1 mL of organoid base medium (step 1.3) on top of the pellet. Vortex the solution at 458 x g, and transfer 300-400 &#181;L to a microcentrifuge tube. Centrifuge at 1,650 x g for 5 min at 4 &#176;C. 
	NOTE: The portion of the cell pellet not placed in the microcentrifuge tube can be frozen down for future use (500 &#181;L of cells to 1 mL of 10% DMSO in FBS) (step 5.1). The frozen cell pellet can be stored for weeks at &#8722;80 &#176;C and for years if placed in liquid nitrogen13.</li>
	<li>Carefully aspirate the organoid base medium using a glass Pasteur pipette, and resuspend in BME using cold tips. 
	NOTE: The amount of BME is based on the size of the pellet. Adding 25% organoid base medium (step 1.3) with resuspended cells to 75% BME is recommended.</li>
	<li>Plate the resuspended cell solution onto a 6-well plate in 40 &#181;L aliquots. Plate up to five aliquots per well.</li>
	<li>Immediately place the well plate in the incubator for 20 min. After incubation, gently add 2 mL of complete organoid medium (step 1.2) into each well.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64878/64878fig02v2.jpg" /> 
Figure 2: Tumor tissue prior to dissection. Representative image of tumor tissue obtained for organoid generation. <a href="https://www.jove.com/files/ftp_upload/64878/64878fig02largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Passaging of organoids
NOTE: If the sample is confluent, each organoid well can be passaged weekly to two new wells.
<ol>
	<li>Utilizing a 1 mL pipette, add 1 mL of organoid base medium (step 1.3) to each well, and pipette the media up and down directly onto the organoid tabs to dissociate it. Collect all the medium containing the resuspended pellets in a 15 mL conical tube.</li>
	<li>Centrifuge the 15 mL conical tube containing the mixture at 1,650 x g for 5 min at 4 &#176;C. Then, carefully aspirate the supernatant using a glass Pasteur pipette.</li>
	<li>Add 1 mL of animal origin-free, recombinant enzyme (see Table of Materials) to the cell pellet, vortex the solution at 458 x g, and transfer to a 1.5 mL microcentrifuge tube. Allow the tube to incubate for 15 min in a 37 &#176;C water bath.</li>
	<li>After incubation, centrifuge at 1,650 x g for 5 min at 4 &#176;C. Then, carefully aspirate the supernatant using a glass Pasteur pipette.</li>
	<li>Resuspend the pellet in BME. 
	NOTE: The amount of BME is based on the size of the pellet. Adding 25% organoid base media (step 1.3) with resuspended cells to 75% BME is recommended.</li>
	<li>Plate the resuspended cell solution into a 6-well plate in 40 &#181;L aliquots. Plate up to five aliquots per well. Once all the aliquots have been plated, immediately place the well plate in the incubator for 20 min. After incubation, gently add 2 mL of complete organoid medium (step 1.2) into each well.</li>
</ol>
5. Freezing and thawing of organoids
<ol>
	<li>Freezing of organoids
	<ol>
		<li>Begin with steps 4.1-4.4.</li>
		<li>Resuspend the cell pellet in 0.5-1 mL of recovery cell culture freezing medium (see Table of Materials), and transfer 1 mL to each cryovial.</li>
		<li>Then, place the cryovials in an isopropanol-filled container at &#8722;80 &#176;C for up to 2 weeks before transferring them to a liquid nitrogen tank for long-term storage13.</li>
	</ol>
	</li>
	<li>Thawing of organoids
	<ol>
		<li>Remove the samples from the liquid nitrogen tank, and thaw at 37 &#176;C water bath.</li>
		<li>Once thawed, transfer to a 15 mL conical tube, and centrifuge at 1,650 x g for 5 min at 4 &#176;C. After centrifugation, carefully aspirate the supernatant using a glass Pasteur pipette.</li>
		<li>Resuspend the pellet in BME. 
		NOTE: The amount of BME is based on the size of the pellet. Adding 25% organoid base media (step 1.3) with resuspended cells to 75% BME is recommended.</li>
		<li>Plate the resuspended cell solution onto a 6-well plate in 40 &#181;L aliquots. Place up to five aliquots per well.</li>
		<li>Immediately place the plate in the incubator for 20 min. After incubation, gently add 2 mL of complete organoid medium (step 1.2) to the wells.</li>
	</ol>
	</li>
</ol>
6. Embedding and generating formalin-fixed paraffin-embedded (FFPE) slides to evaluate the organoid composition
<ol>
	<li>After culturing organoids for at least 10 days to ensure adequate size, remove the complete organoid medium from the wells, and add 1 mL of 2% paraformaldehyde fixative (PFA). Incubate at room temperature for 5-10 min.</li>
	<li>After incubation, wash the cultured organoid aliquots 3x for 5 min each time with 1 mL of PBS.</li>
	<li>Remove the PBS from each well, and add 1 mL of warm 2% agar in deionized H2O to each well. When adding the agar, lift the cultured organoid aliquots from the plate using a spatula. 
	NOTE: It is important not to let the agar harden before lifting the cultured organoid aliquots.
	<ol>
		<li>Allow the agar to solidify in the well at room temperature.</li>
	</ol>
	</li>
	<li>Using a small spatula, free the solidified agar from the well, and store it in a cassette for up to 48 h (see Table of Materials) at 4 &#176;C in 70% ethanol until processing14.</li>
	<li>Embed the samples in paraffin15, cut the slides to a thickness of 5 &#181;m, and stain the slides with hematoxylin and eosin (H&#38;E, see Table of Materials) staining using a standard protocol15.</li>
</ol>

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The authors have nothing to disclose.

Ovarian cancer is extremely deadly due to its advanced stage at diagnosis, as well as the common development of chemotherapy resistance. Many advances in ovarian cancer research have been made by utilizing cancer cell lines and PDX models; however, there is an evident need for a more representative and affordable in vitro model. PDOs have proven to accurately represent the tumor heterogeneity, the tumor microenvironment, and the genomic and transcriptomic features of their primary tumors and, thus, are ideal pre...

In 2021, approximately 21,410 women in the United States were newly diagnosed with epithelial ovarian cancer, and 12,940 women died of this disease1. Although sufficient advancements have been made in surgery and chemotherapy, over 70% of patients with advanced disease develop chemotherapeutic resistance and die within 5 years of diagnosis2,3. Thus, new strategies to treat this deadly disease and representative, reliable models for preclinical research are urgently needed.
Cancer cell lines and patient-derived xenografts (PDX) created from primary ovarian tumors are the main instruments used in ovarian cancer research. A major advantage of cancer cell lines is their rapid expansion. However, their continual culture results in phenotypic and genotypic alterations that cause the cancer cell lines to deviate from the original primary cancer tumor sample. Due to the existing differences between the cancer cell line and the primary tumor, drug assays that have positive effects in cell lines fail to have these same effects in clinical trials2. To overcome these limitations, PDX models are used. These models are created by implanting fresh ovarian cancer tissue into immunodeficient mice. As they are in vivo models, they more accurately resemble human biological characteristics and, in turn, are more predictive of drug outcomes. However, these models also have significant limitations, including the cost, time, and resources needed to generate them4.
PDOs offer an alternative model for preclinical research that overcomes the limitations of both cancer cell lines and PDX models. PDOs recapitulate a patient&#39;s tumor and tumor microenvironment and, thus, provide an in vitro tractable model ideal for preclinical research2,3,5. These 3D models have self-organization capabilities that model the primary tumor, which is a feature that their two-dimensional (2D) cell line counterparts do not possess. Further, these models have been shown to genetically and functionally represent their parent tumors and, thus, are reliable models for studying novel therapeutics and biological processes. In short, they offer long-term expansion and storage capabilities similar to cell lines but also encompass the microenvironment and cell-cell interactions inherent to mouse models4,6.
The present protocol describes the creation of PDOs from patient-derived tumors, ascites, and pleural fluid samples with a higher than 97% success rate. The PDO cultures can then be expanded for multiple generations and used to test drug therapy sensitivity and predictive biomarkers. This method represents a technique that could be used to personalize treatments based on the therapeutic responses of PDOs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1% HEPES</td>
			<td>Life Technologies</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>1% Penicillin-Streptomycin</td>
			<td>Fisher Scientific</td>
			<td>30002CI</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Eppendorf Tubes&#160;</td>
			<td>Genesee Scientific</td>
			<td>14125</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm Tissue Culture Dish&#160;</td>
			<td>TPP</td>
			<td>93100</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Serological Pipet</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#181;m Cell Filter</td>
			<td>MidSci</td>
			<td>100ICS</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>Corning</td>
			<td>430052</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL Cryovial</td>
			<td>Simport Scientific</td>
			<td>T301-2</td>
			<td></td>
		</tr>
		<tr>
			<td>2% Paraformaldehyde Fixative</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#176;C water bath&#160;</td>
			<td>NEST</td>
			<td>602052</td>
			<td></td>
		</tr>
		<tr>
			<td>3dGRO R-Spondin-1 Conditioned Media Supplement</td>
			<td>Millipore Sigma</td>
			<td>SCM104</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plates</td>
			<td>TPP</td>
			<td>92006</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>R31541GA</td>
			<td></td>
		</tr>
		<tr>
			<td>A83-01</td>
			<td>Sigma-Aldrich</td>
			<td>SML0788</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>ThermoFisher</td>
			<td>12634028</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Lamda Biotech</td>
			<td>C121</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex Type 2</td>
			<td>R&#38;D Systems</td>
			<td>3533-010-02</td>
			<td>basement membrane extract</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>New England Bio Labs</td>
			<td>M0303S</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I Reaction Buffer</td>
			<td>New England Bio Labs</td>
			<td>M0303S</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>PeproTech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF-10</td>
			<td>PeproTech</td>
			<td>100-26</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2</td>
			<td>PeproTech</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS C Tubes</td>
			<td>Miltenyi BioTech</td>
			<td>130-096-334</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS Octo Dissociator with Heaters</td>
			<td>Miltenyi BioTech</td>
			<td>130-096-427</td>
			<td>We use the manufacturers protocol.</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Life Technologies</td>
			<td>35050061</td>
			<td>dipeptide, L-alanyl-L-glutamine</td>
		</tr>
		<tr>
			<td>Hematoxylin and Eosin Staining Kit</td>
			<td>Fisher Scientific</td>
			<td>NC1470670</td>
			<td></td>
		</tr>
		<tr>
			<td>Histoplast Paraffin Wax</td>
			<td>Fisher Scientific</td>
			<td>22900700</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mr. Frosty Freezing Container</td>
			<td>Fisher Scientific</td>
			<td>07202363S</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetylcysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636</td>
			<td></td>
		</tr>
		<tr>
			<td>p1000 Pipette with Tips&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>p200 Pipette with Tips&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur Pipettes 9&#34;</td>
			<td>Fisher Scientific</td>
			<td>1367820D</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Fisher Scientific</td>
			<td>MT21031CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet Controller</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prostaglandin E2</td>
			<td>R&#38;D Systems</td>
			<td>2296</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin&#160;</td>
			<td>ThermoFisher</td>
			<td>A1113802</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Murine Noggin</td>
			<td>PeproTech</td>
			<td>250-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Recovery Cell Culture Freezing Medium</td>
			<td>Invitrogen</td>
			<td>12648010</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysis Buffer</td>
			<td>BioLegend</td>
			<td>420301</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK Inhibitor (Y-27632)</td>
			<td>R&#38;D Systems</td>
			<td>1254/1</td>
			<td></td>
		</tr>
		<tr>
			<td>SB202190</td>
			<td>Sigma-Aldrich</td>
			<td>S7076</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 Flask</td>
			<td>MidSci</td>
			<td>TP90076</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Hood&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Embedding Cassette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express</td>
			<td>Invitrogen</td>
			<td>12604013</td>
			<td>animal origin-free, recombinant enzyme</td>
		</tr>
		<tr>
			<td>Type II Collagenase</td>
			<td>Life Technologies</td>
			<td>17101015</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

generation and culturing of high-grade serous ovarian cancer patient-derived organoids

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the discovery of novel therapeutics and predictive biomarkers for ovarian cancer. These models recapitulate clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Additionally, they have been shown to match the primary tumor morphologically, cytologically, immunohistochemically, and genetically. Thus, organoids facilitate research on tumor cells and the tumor microenvironment and are superior to cell lines. The present protocol describes distinct methods to generate patient-derived ovarian cancer organoids from patient tumors, ascites, and pleural fluid samples with a higher than 97% success rate. The patient samples are separated into cellular suspensions by both mechanical and enzymatic digestion. The cells are then plated utilizing a basement membrane extract (BME) and are supported with optimized growth media containing supplements specific to the culturing of high-grade serous ovarian cancer (HGSOC). After forming initial organoids, the PDOs can sustain long-term culture, including passaging for expansion for subsequent experiments.

To generate PDOs, the samples were digested mechanically and enzymatically into single-cell suspensions. The cells were then resuspended in BME and supplemented with specifically engineered media (Figure 3). Organoids are typically established over a time frame of 10 days, after which they demonstrate discrete organoids in culture (Figure 4).
<img alt="Figure 3" class="xfigimg" src="/files/ft...

Watch this Scientific Journal Video about Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids at JoVE.com

Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids

Patient-derived organoids (PDO) are a three-dimensional (3D) culture that can mimic the tumor environment in vitro. In high-grade serous ovarian cancer, PDOs represent a model to study novel biomarkers and therapeutics.

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the ...

generation-culturing-high-grade-serous-ovarian-cancer-patient-derived

Research

JoVE Journal

Cancer Research

4.1K Views.  Washington University in St. Louis. Patient-derived organoids (PDO) are a three-dimensional (3D) culture that can mimic the tumor environment in vitro. In high-grade serous ovarian cancer, PDOs represent a model to study novel biomarkers and therapeutics. 

Video: Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids

High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this clinical challenge, tumor xenograft mouse models using long-established human carcinoma cell lines and many transgenic mouse models with tumors have been developed as preclinical models. They partially mimic the features of human carcinomas, but often fail to recapitulate the key aspects of human malignancies including invasion and metastasis. Thus, alternative models that better represent the malignant progression in human CRC have long been awaited.
We herein show generation of patient-derived tumor xenografts (PDXs) by subcutaneous implantation of small CRC fragments surgically dissected from a patient. The colon PDXs develop and histopathologically resemble the CRC in the patient. However, few spontaneous micrometastases are detectable in conventional cross-sections of affected distant organs in the PDX model. To facilitate the detection of metastatic dissemination into distant organs, we extracted the tumor organoid cells from the colon PDXs in culture and infected them with GFP lentivirus prior to injection into highly immunodeficient NOD/Shi-scid IL2R&#947;null (NOG) mice. Orthotopically injected PDX-derived CRC organoid cells consistently form primary tumors positive for GFP in recipient mice. Moreover, spontaneously developing micrometastatic colonies expressing GFP are notably detected in the lungs of these mice by fluorescence microscopy. Moreover, intrasplenic injection of CRC organoids frequently produces hepatic colonization. Taken together, these findings indicate GFP-labelled PDX-derived CRC organoid cells to be visually detectable during a multistep process termed the invasion-metastasis cascade. The described protocols include the establishment of PDXs of human CRC and 3D culture of the corresponding CRC organoid cells transduced by GFP lentiviral particles.

To allow highly sensitive detection of the disseminating human colorectal cancer (CRC) cells colonizing tissues, we herein show a protocol for efficient transduction of green fluorescent protein (GFP) lentiviral particles into PDX-derived CRC organoid cells prior to their injection into recipient mice, with stereo-fluorescence microscopic observation.

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this ...

A Melanoma Patient-Derived Xenograft Model

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

Patient-derived xenograft (PDX) models more robustly recapitulate melanoma molecular and biological features and are more predictive of therapy response compared to traditional plastic tissue culture-based assays. Here we describe our standard operating protocol for the establishment of new PDX models and the characterization/experimentation of existing PDX models.

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

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.

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 ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are 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) ...

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture models. PDOs have been successfully generated from a variety of human tumors to recapitulate the architecture and function of tumor tissue accurately and efficiently. However, PDOs are unsuitable for an in vitro high-throughput assay system (HTS) or cell analysis using 96-well or 384-well plates when evaluating anticancer drugs because they are heterogeneous in size and form large clusters in culture. These cultures and assays use extracellular matrices, such as Matrigel, to create tumor tissue scaffolds. Therefore, PDOs have a low throughput and high cost, and it has been difficult to develop a suitable assay system. To address this issue, a simpler and more accurate HTS was established using PDOs to evaluate the potency of anticancer drugs and immunotherapy. An in vitro HTS was created that uses PDOs established from solid tumors cultured in 384-well plates. An HTS was also developed for assessment of antibody-dependent cellular cytotoxicity activity to represent the immune response using PDOs cultured in 96-well plates.

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

A highly accurate in vitro high-throughput assay system was developed to evaluate anticancer drugs using patient-derived tumor organoids (PDOs), similar to cancer tissues but are unsuitable for in vitro high-throughput assay systems with 96-well and 384-well plates.

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture ...

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and treatment response to targeted inhibitors in cancer. Pioneering work in gastrointestinal and pancreatic cancers has highlighted the promise of patient-derived organoids (PDOs) as a patient-proximate culture system, with an increasing number of models emerging. Similarly, work in other cancer types has focused on establishing organoid models and optimizing culture protocols. Notably, 3D cancer organoid models maintain the genetic complexity of original tumor specimens and thus translate tumor-derived sequencing data into treatment with genetically informed targeted therapies in an experimental setting. Further, PDOs might foster the evaluation of rational combination treatments to overcome resistance-associated adaptation of tumors in the future. The latter focuses on intense research efforts in non-small-cell lung cancer (NSCLC), as resistance development ultimately limits the treatment success of targeted inhibitors. An early assessment of therapeutically targetable mechanisms using NSCLC PDOs could help inform rational combination treatments. This manuscript describes a standardized protocol for the cell culture plate-based assessment of drug sensitivities to targeted inhibitors in NSCLC-derived 3D PDOs, with potential adaptability to combinational treatments and other treatment modalities.

This protocol describes a standardized evaluation of drug sensitivities to targeted signaling inhibitors in NSCLC patient-derived organoid models.

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and ...

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D tumoroids. Since patient derived tumor organoids can retain the characteristics of the original tumor, these reliable preclinical models enable cancer drug screening and the study of drug resistance mechanisms. However, CRC related death in patients is mostly associated with the presence of metastatic disease. It is therefore essential to evaluate the efficacy of anti-cancer therapies in relevant in vivo models that truly recapitulate the key molecular features of human cancer metastasis. We have established an orthotopic model based on the injection of CRC patient-derived cancer cells directly into the cecum wall of mice. These tumor cells develop primary tumors in the cecum that metastasize to the liver and lungs, which is frequently observed in patients with advanced CRC. This CRC mouse model can be used to evaluate drug responses monitored by microcomputed tomography (&#181;CT), a clinically relevant small-scale imaging method that can easily identify primary tumors or metastases in patients. Here, we describe the surgical procedure and the required methodology to implant patient-derived cancer cells in the cecum wall of immunodeficient mice.

This protocol describes the orthotopic implantation of patient-derived cancer cells in the cecum wall of immunodeficient mice. The model recapitulates advanced colorectal cancer metastatic disease and allows for the evaluation of new therapeutic drugs in a clinically relevant scenario of lung and liver metastases.

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D ...

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 ...

Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids

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Rozdziały w tym wideo

High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor

A Melanoma Patient-Derived Xenograft Model

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

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

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

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

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

Establishment and Culture of Patient-Derived Breast Organoids