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 µmol/L prostaglandin E2, 10 µmol/L SB202190, 500 nmol/L A83-01, and 10 µM ROCK inhibitor (see Table of Materials). 
	​NOTE: The medium may be stored at 4 °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 °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 °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 µg/mL DNase I solution by combining 1,000 µL of nuclease-free water, 100 µL of DNase I reaction buffer, and 10 µL 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 µg/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 °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 °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 °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 µL to a microcentrifuge tube. Centrifuge the microcentrifuge tube at 1,650 x g for 5 min at 4 °C. 
	NOTE: The portion of the cell pellet not placed in the microcentrifuge tube can be frozen down for future use (500 µL of cells to 1 mL of 10% DMSO in FBS). The frozen cell pellet can be stored for weeks at −80 °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 µL 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 °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="Lab pipetting setup, molecular biology experiment, cell culture preparation, precise liquid handling." class="xfigimg" src="/files/ftp_upload/64878/64878fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
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 µm 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 °C. Then, carefully aspirate the supernatant using a glass Pasteur pipette.</li>
	<li>Prepare a 100 µg/mL DNase I solution by combining 1,000 µL of nuclease-free water, 100 µL of DNase I reaction buffer, and 10 µL 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 °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 °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 °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 µL to a microcentrifuge tube. Centrifuge at 1,650 x g for 5 min at 4 °C. 
	NOTE: The portion of the cell pellet not placed in the microcentrifuge tube can be frozen down for future use (500 µL of cells to 1 mL of 10% DMSO in FBS) (step 5.1). The frozen cell pellet can be stored for weeks at −80 °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 µL 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="Cell culture experiment setup with media and petri dish for tissue growth analysis." class="xfigimg" src="/files/ftp_upload/64878/64878fig02v2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
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 °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 °C water bath.</li>
	<li>After incubation, centrifuge at 1,650 x g for 5 min at 4 °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 µL 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 −80 °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 °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 °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 µL 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 °C in 70% ethanol until processing14.</li>
	<li>Embed the samples in paraffin15, cut the slides to a thickness of 5 µm, and stain the slides with hematoxylin and eosin (H&amp;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><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&amp;nbsp;</td><td>Genesee Scientific</td><td>14125</td><td></td></tr><tr><td>10 cm Tissue Culture Dish&amp;nbsp;</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 &amp;micro;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 &amp;deg;C water bath&amp;nbsp;</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&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Cultrex Type 2</td><td>R&amp;amp;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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>p200 Pipette with Tips&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Pasteur Pipettes 9&quot;</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&amp;amp;D Systems</td><td>2296</td><td></td></tr><tr><td>Puromycin&amp;nbsp;</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&amp;amp;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&amp;nbsp;</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="Tumor organoid formation diagram; patien...

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 ...

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Research

JoVE Journal

Cancer Research

4.2K Views.  Washington University in St. Louis. Patiëntgestuurde organoïde modellen vertegenwoordigen genetisch en functioneel hun oudertumoren en zijn dus betrouwbare modellen voor het bestuderen van nieuwe therapieën en complexe biologische processen bij eierstokkanker. Door klonale heterogeniteit, de micro-omgeving van de tumor en in vitro cel-tot-celinteracties samen te vatten, overwinnen deze modellen de beperkingen van kankercellijnen en van patiënten afgeleide xenografts. Van de patiënt afgeleide organoïden bieden langdurige u...

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. ...

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 ...

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

Immunofluorescence is one of the most widely used techniques to visualize target antigens with high sensitivity and specificity, allowing for the accurate identification and localization of proteins, glycans, and small molecules. While this technique is well-established in two-dimensional (2D) cell culture, less is known about its use in three-dimensional (3D) cell models. Ovarian cancer organoids are 3D tumor models that recapitulate tumor cell clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Thus, they are superior to cell lines for the evaluation of drug sensitivity and functional biomarkers. Therefore, the ability to utilize immunofluorescence on primary ovarian cancer organoids is extremely beneficial in understanding the biology of this cancer. The current study describes the technique of immunofluorescence to detect DNA damage repair proteins in high-grade serous patient-derived ovarian cancer organoids (PDOs). After exposing the PDOs to ionizing radiation, immunofluorescence is performed on intact organoids to evaluate nuclear proteins as foci. Images are collected using z-stack imaging on confocal microscopy and analyzed using automated foci counting software. The described methods allow for the analysis of temporal and special recruitment of DNA damage repair proteins and colocalization of these proteins with cell-cycle markers.

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

The present protocol describes methods for evaluating DNA damage repair proteins in patient-derived ovarian cancer organoids. Included here are comprehensive plating and staining methods, as well as detailed, objective quantification procedures.

Immunofluorescence is one of the most widely used techniques to visualize target antigens with high sensitivity and specificity, allowing for the accurate ...

Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts from Fresh Tissue

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

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

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

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

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

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 ...

Derivation of 3D Glioma Organoids from Chopper-Processed Patient Tumors

Cultivating Ex Vivo Patient-Derived Glioma Organoids Using a Tissue Chopper

Glioblastoma, IDH-wild type, CNS WHO grade 4 (GBM) is a primary brain tumor associated with poor patient survival despite aggressive treatment. Developing realistic ex vivo models remain challenging. Patient-derived 3-dimensional organoid (PDO) models offer innovative platforms that capture the phenotypic and molecular heterogeneity of GBM, while preserving key characteristics of the original tumors. However, manual dissection for PDO generation is time-consuming, expensive and can result in a number of irregular and unevenly sized PDOs. This study presents an innovative method for PDO production using an automated tissue chopper. Tumor samples from four GBM and one astrocytoma, IDH-mutant, CNS WHO grade 2 patients were processed manually as well as using the tissue chopper. In the manual approach, the tumor material was dissected using scalpels under microscopic control, while the tissue chopper was employed at three different angles. Following culture on an orbital shaker at 37 &#176;C, morphological changes were evaluated using bright field microscopy, while proliferation (Ki67) and apoptosis (CC3) were assessed by immunofluorescence after 6 weeks. The tissue chopper method reduced almost 70% of the manufacturing time and resulted in a significantly higher PDOs mean count compared to the manually processed tissue from the second week onwards (week 2: 801 vs. 601, P = 0.018; week 3: 1105 vs. 771, P = 0.032; and week 4:1195 vs. 784, P &lt; 0.01). Quality assessment revealed similar rates of tumor-cell apoptosis and proliferation for both manufacturing methods. Therefore, the automated tissue chopper method offers a more efficient approach in terms of time and PDO yield. This method holds promise for drug- or immunotherapy-screening of GBM patients.

Author Spotlight: Enhanced Generation of Patient-Derived 3D Organoids for Glioblastoma and Glioma

This study introduces an automated method to generate patient-derived glioblastoma 3-dimensional organoids utilizing a tissue chopper. The method provides a suitable and effective approach to obtain such organoids for therapeutical testing.

Glioblastoma, IDH-wild type, CNS WHO grade 4 (GBM) is a primary brain tumor associated with poor patient survival despite aggressive treatment. Developing ...

Standardizing Ovarian Cancer Organoid Biobanking

Strategy for Biobanking of Ovarian Cancer Organoids: Addressing the Interpatient Heterogeneity across Histological Subtypes and Disease Stages

While the establishment of an ovarian cancer biobank from patient-derived organoids along with their clinical background information promises advances in research and patient care, standardization remains a challenge due to the heterogeneity of this lethal malignancy, combined with the inherent complexity of organoid technology. This adaptable protocol provides a systematic framework to realize the full potential of ovarian cancer organoids considering a patient-specific variability of progenitors. By implementing a structured experimental workflow to select optimal culture conditions and seeding methods, with parallel testing of direct 3D seeding versus a 2D/3D route, we obtain, in most cases, robust long-term expanding lines suitable for a broad range of downstream applications. 
Notably, the protocol has been tested and proven efficient in a great number of cases (N = 120) of highly heterogeneous starting material, including high-grade and low-grade ovarian cancer and stages of the disease with primary debulking, recurrent disease, and post-neoadjuvant surgical specimens. Within a low Wnt, high BMP exogenous signaling environment, we observed progenitors being differently susceptible to the activation of the Heregulin 1 &#223; (HER&#223;-1)-pathway, with HER&#223;-1 promoting organoid formation in some while inhibiting it in others. For a subset of the patient's samples, optimal organoid formation and long-term growth necessitate the addition of fibroblast growth factor 10 and R-Spondin 1 to the medium. 
Further, we highlight the critical steps of tissue digestion and progenitor isolation and point to examples where brief cultivation in 2D on plastic is beneficial for subsequent organoid formation in the Basement Membrane Extract type 2 matrix. Overall, optimal biobanking requires systematic testing of all main conditions in parallel to identify an adequate growth environment for individual lines. The protocol also describes the handling procedure for efficient embedding, sectioning, and staining to obtain high-resolution images of organoids, which is required for comprehensive phenotyping.

Author Spotlight: Advancing Personalized Medicine in Ovarian Cancer 

This protocol offers a systematic framework for the establishment of ovarian cancer organoids from different disease stages and addresses the challenges of patient-specific variability to increase yield and enable robust long-term expansion for subsequent applications. It includes detailed steps for tissue processing, seeding, adjusting media requirements, and immunofluorescence staining.

While the establishment of an ovarian cancer biobank from patient-derived organoids along with their clinical background information promises advances in ...

Human Ovarian Epithelium Organoids: A Model for Tissue Repair and Drug Testing

Human Ovarian Surface Epithelium Organoids as a Platform to Study Tissue Regeneration

The ovarian surface epithelium (OSE), the outermost layer of the ovary, undergoes rupture during each ovulation and plays a crucial role in ovarian wound healing while restoring ovarian integrity. Additionally, the OSE may serve as the source of epithelial ovarian cancers. Although the OSE regenerative properties have been well studied in mice, understanding the precise mechanism of tissue repair in the human ovary remains hampered by limited access to human ovaries and suitable in vitro culture protocols. Tissue-specific organoids, miniaturized in vitro models replicating both structural and functional aspects of the original organ, offer new opportunities for studying organ physiology, disease modeling, and drug testing. 
Here, we describe a method to isolate primary human OSE (hOSE) from whole ovaries and establish hOSE organoids. We include a morphological and cellular characterization showing heterogeneity between donors. Additionally, we demonstrate the capacity of this culture method to evaluate hormonal effects on OSE-organoid growth over a 2-week period. This method may enable the discovery of factors contributing to OSE regeneration and facilitate patient-specific drug screenings for malignant OSE.

Author Spotlight: Advancing Human Ovarian Repair and Cancer Studies with Surface Epithelium Organoids

This protocol describes establishing three-dimensional (3D) tissue organoids from primary human ovarian surface epithelium (hOSE) cells. The protocol includes isolation of hOSE from freshly collected ovaries, cellular expansion of the hOSE, cryopreservation-thawing procedures, and organoid derivation. Immunofluorescence, quantitative analysis, and showcasing utility as a screening platform are included.

The ovarian surface epithelium (OSE), the outermost layer of the ovary, undergoes rupture during each ovulation and plays a crucial role in ovarian wound ...

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

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