Name: generation and culturing of high-grade serous ovarian cancer patient-derived organoids
Uploaded: 2023-01-06T14:50:00
Description: Watch this Scientific Journal Video about Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids at JoVE.com

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 ...

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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.

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			<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>
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			<td>1.5 mL Eppendorf Tubes&#160;</td>
			<td>Genesee Scientific</td>
			<td>14125</td>
			<td></td>
		</tr>
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			<td>10 cm Tissue Culture Dish&#160;</td>
			<td>TPP</td>
			<td>93100</td>
			<td></td>
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			<td>10 mL Serological Pipet</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
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			<td>100 &#181;m Cell Filter</td>
			<td>MidSci</td>
			<td>100ICS</td>
			<td></td>
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		<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>
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			<td>37 &#176;C water bath&#160;</td>
			<td>NEST</td>
			<td>602052</td>
			<td></td>
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			<td>3dGRO R-Spondin-1 Conditioned Media Supplement</td>
			<td>Millipore Sigma</td>
			<td>SCM104</td>
			<td></td>
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		<tr>
			<td>6 well plates</td>
			<td>TPP</td>
			<td>92006</td>
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			<td>70% Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>R31541GA</td>
			<td></td>
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			<td>A83-01</td>
			<td>Sigma-Aldrich</td>
			<td>SML0788</td>
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			<td>Advanced DMEM/F12</td>
			<td>ThermoFisher</td>
			<td>12634028</td>
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			<td>Agar</td>
			<td>Lamda Biotech</td>
			<td>C121</td>
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			<td>B-27</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td></td>
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			<td>Centrifuge&#160;</td>
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			<td></td>
			<td></td>
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			<td>Cultrex Type 2</td>
			<td>R&#38;D Systems</td>
			<td>3533-010-02</td>
			<td>basement membrane extract</td>
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		<tr>
			<td>DNase I</td>
			<td>New England Bio Labs</td>
			<td>M0303S</td>
			<td></td>
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			<td>DNase I Reaction Buffer</td>
			<td>New England Bio Labs</td>
			<td>M0303S</td>
			<td></td>
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			<td>EGF</td>
			<td>PeproTech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
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			<td>FBS&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td></td>
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			<td>FGF-10</td>
			<td>PeproTech</td>
			<td>100-26</td>
			<td></td>
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			<td>FGF2</td>
			<td>PeproTech</td>
			<td>100-18B</td>
			<td></td>
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			<td>gentleMACS C Tubes</td>
			<td>Miltenyi BioTech</td>
			<td>130-096-334</td>
			<td></td>
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			<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>
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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 ...

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