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Cancer Research

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

Published: January 6th, 2023

DOI:

10.3791/64878

1Washington University in St. Louis, 2University of California San Francisco
* These authors contributed equally

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 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.

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 tum....

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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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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).

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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.......

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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's School of Medicine in St. Louis's Department of Obstetrics and Gynecology and Division of Gynecologic Oncology, Washington University's Dean's Scholar Program, and the Reproductive Scientist Development Program for their support of this project.

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NameCompanyCatalog NumberComments
1% HEPESLife Technologies15630080
1% Penicillin-StreptomycinFisher Scientific30002CI
1.5 mL Eppendorf Tubes Genesee Scientific14125
10 cm Tissue Culture Dish TPP93100
10 mL Serological Pipet
100 µm Cell FilterMidSci100ICS
15 mL centrifuge tubesCorning430052
2 mL CryovialSimport ScientificT301-2
2% Paraformaldehyde FixativeSigma-Aldrich
37 °C water bath NEST602052
3dGRO R-Spondin-1 Conditioned Media SupplementMillipore SigmaSCM104
6 well platesTPP92006
70% EthanolSigma-AldrichR31541GA
A83-01Sigma-AldrichSML0788
Advanced DMEM/F12ThermoFisher12634028
AgarLamda BiotechC121
B-27Life Technologies17504044
Centrifuge 
Cultrex Type 2R&D Systems3533-010-02basement membrane extract
DNase INew England Bio LabsM0303S
DNase I Reaction BufferNew England Bio LabsM0303S
EGFPeproTechAF-100-15
FBS Sigma-AldrichF2442
FGF-10PeproTech100-26
FGF2PeproTech100-18B
gentleMACS C TubesMiltenyi BioTech130-096-334
gentleMACS Octo Dissociator with HeatersMiltenyi BioTech130-096-427We use the manufacturers protocol.
GlutaMAXLife Technologies35050061dipeptide, L-alanyl-L-glutamine
Hematoxylin and Eosin Staining KitFisher ScientificNC1470670
Histoplast Paraffin WaxFisher Scientific22900700
Microcentrifuge 
Mr. Frosty Freezing ContainerFisher Scientific07202363S
N-acetylcysteineSigma-AldrichA9165
NicotinamideSigma-AldrichN0636
p1000 Pipette with Tips 
p200 Pipette with Tips 
Pasteur Pipettes 9"Fisher Scientific1367820D
PBSFisher ScientificMT21031CM
Pipet Controller
Prostaglandin E2R&D Systems2296
Puromycin ThermoFisherA1113802
Recombinant Murine NogginPeproTech250-38
Recovery Cell Culture Freezing MediumInvitrogen12648010
Red Blood Cell Lysis BufferBioLegend420301
ROCK Inhibitor (Y-27632)R&D Systems1254/1
SB202190Sigma-AldrichS7076
T75 FlaskMidSciTP90076
Tissue Culture Hood 
Tissue Embedding Cassette
TrypLE ExpressInvitrogen12604013animal origin-free, recombinant enzyme
Type II CollagenaseLife Technologies17101015
Vortex

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