We would like to thank the staff of the Tissue Procurement Facility of the University of Minnesota for assistance with patient tissue samples collection. This work was supported by the Department of Defense Ovarian Cancer Research Program (OCRP) OC093424 to MB, by the Randy Shaver Cancer Research and Community Fund to MB, by the Minnesota Ovarian Cancer Alliance to MB and by the Gynecological Oncology Departmental fund to MB. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Ethics Statement&#160; 
Solid specimens of ovarian cancer were obtained from the University of Minnesota Tissue Procurement Facility (TPF) after Institutional Review Board Committee: Human Subject Board (IRB) approval.
1. Reagent Setup
<ol>
	<li>Prepare Complete DMEM medium by supplementing DMEM with 10% FBS, and 100 units penicillin-streptomycin. Store the medium at 4&#160;&#176;C and warm to 37&#160;&#176;C prior to use.</li>
	<li>Aliquot dispase II into separate volumes of 5-10 ml to avoid multiple freeze thaws and store at -20&#160;&#176;C in a nonfrost-free freezer.</li>
</ol>
2. Tissue Collection
<ol>
	<li>Collect solid specimens of ovarian cancer (~5 g in size) at the time of surgery from areas macroscopically identified as cancer by a pathologist. Clinical specimens are de-identified and registered according to institutional protocols.</li>
	<li>Place specimens in a sterile, screw lid, polypropylene specimens container filled with 30 ml of ice-cold PBS. Transport the specimens from the surgical pathology laboratory to the research laboratory for processing on ice, and within 30 min&#160;from specimen collection&#160;(Figure 1).</li>
</ol>
3. Tissue Processing
<ol>
	<li>Working under sterile conditions, transfer the samples onto a Petri dish (60 mm x&#160;60 mm) containing 10 ml of fresh, ice-cold PBS and using a sterile razor blade, further cut into the smallest pieces possible (2 mm or less)&#160;(Figures 2A and 2B).</li>
	<li>Transfer the minced tissues into a 15 ml conic tube containing 10 ml of prewarmed (37 &#176;C for 30 min) dispase II (2.4 U/ml) in DMEM and incubate at 5% CO2 and 37 &#176;C for 30 min. To ensure optimal digestion of the specimens, manually agitate the cell slurry every 5 min.</li>
	<li>After 30 min&#160;incubation, transfer (using a 10 ml serological pipette) the cell slurry onto a cell strainer&#160;(70 &#956;m mesh) placed on top of a 50 ml conical tube and apply a gentle pressure against the mesh using a syringe plunger. Discard any undissociated tissue (remaining on the top of the mesh) and collect the obtained cell suspension in the 50 ml sterile conical tube. Centrifuge at 320 x g for 7 min&#160;at 4 &#176;C&#160;(Figure 3).</li>
	<li>Discard the supernatant and resuspend the cell pellet in 10 ml of DMEM containing 10% FBS.</li>
	<li>Incubate the cell suspension in a Petri dish at 5% CO2 and 37 &#176;C&#160;(Figure 4).</li>
	<li>Change the medium after 24 hr&#160;from the initial plating. This allows for removal of cellular debris and the majority of the erythrocytes present in culture.</li>
	<li>Change the medium every three days for the following two weeks, after which the cultures of primary EOC are ready for downstream applications.</li>
</ol>

<ol><li>Kurman, R. J., Visvanathan, K., Roden, R., Wu, T. C., Ie M, S. hih <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+detection+and+treatment+of+ovarian+cancer%3A+shifting+from+early+stage+to+minimal+volume+of+disease+based+on+a+new+model+of+carcinogenesis%5BTitle%5D)+AND+%22Am.+J.+Obstet.+Gynecol%22%5BJournal%5D)">Early detection and treatment of ovarian cancer: shifting from early stage to minimal volume of disease based on a new model of carcinogenesis.</a> Am. J. Obstet. Gynecol. 198 (4), 351-356 (2008).</li>
<li>Kuhn, E., Kurman, R. J., Shih, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ovarian+Cancer+Is+an+Imported+Disease%3A+Fact+or+Fiction%5BTitle%5D)+AND+%22Curr.+Obstet.+Gynecol.+Rep%22%5BJournal%5D)">Ovarian Cancer Is an Imported Disease: Fact or Fiction.</a> Curr. Obstet. Gynecol. Rep. 1 (1), 1-9 (2012).</li>
<li>Sarojini, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+detection+biomarkers+for+ovarian%5BTitle%5D)+AND+%22J.+Oncol%22%5BJournal%5D)">Early detection biomarkers for ovarian.</a> J. Oncol. , (2012).</li>
<li>Shepherd, T. G., Theriault, B. L., Campbell, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nachtigal+M.W.+Primary+culture+of+ovarian+surface+epithelial+cells+and+ascites-derived+ovarian+cancer+cells+from+patients%5BTitle%5D)+AND+%22Nat.+Protoc%22%5BJournal%5D)">Nachtigal M.W. Primary culture of ovarian surface epithelial cells and ascites-derived ovarian cancer cells from patients.</a> Nat. Protoc. 1 (6), 2643-2649 (2006).</li>
<li>Tsao, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+human+ovarian+surface+epithelial+cells+immortalized+by+human+papilloma+viral+oncogenes+%28HPV-E6E7+ORFs%29%5BTitle%5D)+AND+%22Exp.+Cell.+Res%22%5BJournal%5D)">Characterization of human ovarian surface epithelial cells immortalized by human papilloma viral oncogenes (HPV-E6E7 ORFs).</a> Exp. Cell. Res. 218 (2), 499-507 (1995).</li>
<li>Auersperg, N., Maines-Bandiera, S. L., Dyck, H. G., Kruk, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+cultured+human+ovarian+surface+epithelial+cells%3A+phenotypic+plasticity+and+premalignant+changes%5BTitle%5D)+AND+%22Lab.+Invest%22%5BJournal%5D)">Characterization of cultured human ovarian surface epithelial cells: phenotypic plasticity and premalignant changes.</a> Lab. Invest. 71 (4), 510-518 (1994).</li>
<li>Brewer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+model+of+normal%2C+immortalized+ovarian+surface+epithelial+and+ovarian+cancer+cells+for+chemoprevention+of+ovarian+cancer%5BTitle%5D)+AND+%22Gynecol.+Oncol%22%5BJournal%5D)">In vitro model of normal, immortalized ovarian surface epithelial and ovarian cancer cells for chemoprevention of ovarian cancer.</a> Gynecol. Oncol. 98 (2), 182-192 (2005).</li>
<li>Sueblinvong, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishment%2C+characterization+and+downstream+application+of+primary+ovarian+cancer+cells+derived+from+solid+tumors%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Establishment, characterization and downstream application of primary ovarian cancer cells derived from solid tumors.</a> PLoS One. 7 (11), (2012).</li>
<li>Rockwell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo-in+vitro+tumour+cell+lines%3A+characteristics+and+limitations+as+models+for+human+cancer%5BTitle%5D)+AND+%22Br.+J.+Cancer+Suppl%22%5BJournal%5D)">In vivo-in vitro tumour cell lines: characteristics and limitations as models for human cancer.</a> Br. J. Cancer Suppl. 41 (4), 118-122 (1980).</li>
<li>Wong, C., Chen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+development%2C+application+and+limitations+of+breast+cancer+cell+lines+to+study+tamoxifen+and+aromatase+inhibitor+resistance%5BTitle%5D)+AND+%22J.+Steroid.+Biochem.+Mol.+Biol%22%5BJournal%5D)">The development, application and limitations of breast cancer cell lines to study tamoxifen and aromatase inhibitor resistance.</a> J. Steroid. Biochem. Mol. Biol. 131 (3-5), 83-92 (2012).</li>
</ol>

The authors have nothing to disclose.

A better understanding of ovarian cancer etiology and development is crucial to improving the outcome of women affected by this devastating disease. In this context, the use of established and &#34;commercially available&#34; ovarian cancer cell line has undoubtedly been tremendously useful. However, today we know that cancer cell lines are not representative of the human tumor they originated from in many aspects including the lack of heterogeneity9,10.
Here, we report a rapid and reliable method for isolation and culturing of primary ovarian cancer cells directly from clinical specimens of ovarian cancer within 30 min&#160;of surgical specimen isolation.
Because the primary cells are isolated from a solid specimen versus ascites fluid, this has the advantage of isolating cells from early stages of the disease prior to the time when ascites fluid formation occurs. This technique also has the advantage of isolating primary ovarian cancer cells from a specific site. The success in obtaining viable primary ovarian cancer cells using this protocol is largely dependent on how quickly the sample can be processed following surgery. For best results, the specimens should be received within 30 min&#160;from surgery. If this is not possible, the specimen should be kept at 4&#160;&#176;C (in PBS) overnight and processed the following day. In our experience, processing of samples following overnight storage decreases the yield.&#160;Exposure to dispase II for longer than 30 min&#160;also reduces cell viability and should not be performed. An alternative to dispase II treatment can be treatment with collagenase or hyaluronidase. However, this results in decreased yield8.&#160; Because of the great variability in clinical specimens, some may have larger red blood cell contamination as compared to others. If that is the case, we suggest &#34;washing&#34; the minced tissues (step 3.1) with PBS prior exposing them to enzymatic digestion. This will reduce the number of red blood cells in the preparation and ultimately facilitate adhesion of the primary epithelial cells.
Because the clinical specimens are not sterile (they lose sterility as soon as they arrive at the surgical pathology laboratory), it is critical to perform all of the steps under strictly sterile conditions. While contamination of the culture is possible, typically it occurs in less than 10% of the cases.&#160;The above protocol may be used with any consistency of clinical specimen.
The major limitation of this protocol is that the number of cells recovered from each samples is highly dependent on the size of the specimens provided. A typical specimen&#39;s size is about 3 cm x 3 cm. Another limitation is represented by the fact that while primary ovarian cancer cells grow exponentially for up to 10 passages in vitro (therefore providing the opportunity to make viable stocks) they ultimately senesce and die, thus limiting the availability of cells from a single donor.&#160;While we do not perform any separation of tumor cells from fibroblasts, we hypothesize that the relatively high (10%) concentration of serum throughout the process may give ovarian cancer cells a survival advantage over fibroblasts. It is also likely that cells that are less prone to separation from the stromal compartment (fibroblasts) may not disaggregate and will remain on the filter during separation.
Cells obtained with this protocol are highly suitable for future applications including investigations aimed to understand the processes leading to ovarian cancer initiation and development. Future applications also include using these primary cells for in vitro and in vivo testing of novel chemotherapy agents for ovarian cancer as well as biomarkers for early detection.

Despite its relatively low incidence, ovarian cancer is the deadliest of the gynecological diseases and the fifth leading cause of cancer deaths among women1,2. This is mainly due to the lack of reliable tools and models that faithfully recapitulate the initiation and progression of the disease3. Most of our knowledge today about ovarian cancer has been possible through the use of immortalized ovarian surface epithelial cells (IOSEs), ovarian cancer cell lines and primary ovarian cancer cells recovered from ascitic fluid4-7. Unfortunately, their use has several limitations including a number of genetic and phenotypic changes associated with immortalization process or in vitro passages and the heterogeneity of the population resulting from ascitic fluid preparation.
Therefore, primary ovarian cancer cells derived from identifiable and specific solid specimens of ovarian cancer represent a unique tool for studying ovarian cancer progression.
The main difficulties involved in the obtaining these malignant cells are due to an overgrowth of stromal cells or fibroblasts along with loss of viability and premature lack of proliferative capacity in the culture of these EOC cells. Several methods to create single-cell suspensions from solid tumors currently exist, through mechanical means or enzymatic dissociation, however certain techniques yield a greater amount of the preferred outcome8. Here, we show that enzymatic digestion with dispase II results in an effective recovery of viable, fibroblast-free EOC cells. The so-obtained EOC cultures are highly susceptible to genetic manipulation and are also useful in drug screening tests, indicating that these EOC cultures are suitable for many downstream applications.

<table><tbody><tr><td>1x PBS, sterile&amp;nbsp;</td><td>Invitrogen</td><td>14190-144</td><td></td></tr><tr><td>Screw lid, polypropylene specimen container, sterile</td><td>Thermo Scientific</td><td>02 1090</td><td></td></tr><tr><td>DMEM medium, sterile&amp;nbsp;</td><td>Invitrogen</td><td>11995-065</td><td></td></tr><tr><td>Fetal bovine serum, sterile</td><td>Thermo Scientific Hyclone</td><td>SH30396.03</td><td></td></tr><tr><td>100x Penicillin-streptomycin, liquid</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>Dispase II, sterile</td><td>Roche</td><td>04942 078 001</td><td></td></tr><tr><td>Small fine-tip forceps and scalpel blade, sterile</td><td>Fisher Scientific</td><td>forceps: 08-953E, blades: 08-927-5D</td><td></td></tr><tr><td>Small dissecting scissors, sterile</td><td>Fisher Scientific</td><td>08-945</td><td></td></tr><tr><td>15 ml Conical capped tubes, sterile</td><td>BD Falcon</td><td>352097</td><td></td></tr><tr><td>50 ml Conical capped tubes, sterile</td><td>BD Falcon</td><td>352027</td><td></td></tr><tr><td>10 ml Serological pipette, sterile&amp;nbsp;</td><td>Corning</td><td>13-678-11E</td><td></td></tr><tr><td>6 ml Syringe plunger, sterile&amp;nbsp;</td><td>Tyco Healthcare</td><td>8881516911</td><td></td></tr><tr><td>Nylon filter (70 &amp;mu;m ) cell strainer, sterile</td><td>BD Falcon</td><td>352350</td><td></td></tr><tr><td>5 cm Petri dish, sterile&amp;nbsp;</td><td>Corning</td><td>430166</td><td></td></tr><tr><td>10 cm Petri dish, sterile&amp;nbsp;</td><td>Corning</td><td>430167</td><td></td></tr></tbody></table>

method for obtaining primary ovarian cancer cells from solid specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

Fresh clinical specimens of ovarian cancer are collected after surgery (Figure 1) and cut in small pieces (Figures 2A and 2B) constituting the cell slurry. This allows for optimal exposure of the specimens to the enzymatic treatment. Cell slurry is exposed to enzymatic digestion and incubated at 37 °C for 30 min. During the incubation time the slurry becomes increasingly cloudy (especially after each agitation) and this is a sign of tissue disaggregation. At the end of the incubation time, the slurry is transferred onto a cell strainer to separate EOC cells from any undissociated tissue (Figure 3). The recovered cell suspension is placed at 5% CO2 and 37 °C (Figure 4). At this stage the morphology of the cell cultures reveals the presence of both EOC cells as a single cell suspension and in small clumps. The culture at this point also reveals the presence of erythrocytes and small debris as shown in Figure 5. Importantly while the EOC will slowly (over a period of 1-3 days) adhere to the plastic, erythrocytes and cell debris will not and they will eventually and progressively "disappear from the culture". By day 3 from initial plating, the EOC cultures show increasing adherent EOC cell clusters and progressively less erythrocyte and debris as shown in Figure 6. By day 6-7, EOC cells form swirl-like shapes (arrow) and start to spread to the plastic to form larger multicellular aggregates leading to the typical cobblestone morphology of EOC cells as shown in Figure 7. By day 14, EOC cells typically become confluent (Figure 8) and are now ready for downstream testing and applications.
<img alt="Sample collection container with labeled specimen for histological analysis, laboratory setup." fo:content-width="5in" fo:src="/files/ftp_upload/51581/51581fig1highres.jpg" src="/files/ftp_upload/51581/51581fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Collection of clinical specimens. Solid specimens of ovarian cancer are collected at the time of surgery and placed in a sterile, screw lid, polypropylene specimen container filled with 30 ml of ice-cold PBS.
<img alt="Tissue dissection in petri dish; laboratory experiment setup; sample preparation process." fo:content-width="5in" fo:src="/files/ftp_upload/51581/51581fig2ahighres.jpg" src="/files/ftp_upload/51581/51581fig2a.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2a">
<img alt="Petri dish with plant tissue culture in sterile lab; plant regeneration experiment setup." fo:content-width="5in" fo:src="/files/ftp_upload/51581/51581fig2bhighres.jpg" src="/files/ftp_upload/51581/51581fig2b.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2b"> 
Figure 2. Processing of clinical specimens. A) Solid specimen transferred onto a Petri dish containing 10 ml of fresh, ice-cold 1x PBS. B) Clinical specimens further cut into pieces about 2 mm in size.
<img alt="Lab chromatography setup for protein purification using syringe and filter; hands manipulating syringe." fo:content-width="5in" fo:src="/files/ftp_upload/51581/51581fig3highres.jpg" src="/files/ftp_upload/51581/51581fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. Separation of EOC cells from undissociated tissues. Following enzymatic digestion, clinical specimens are transferred onto a cell strainer and a gentle pressure is applied on the digested clinical specimens by using a syringe plunger. This allow for mechanical detachment of EOC from connective tissues and recovery of EOC cells.
<img alt="Cell culture incubation with Petri dishes, microbiology experiment setup, cell growth analysis." fo:content-width="5in" fo:src="/files/ftp_upload/51581/51581fig4highres.jpg" src="/files/ftp_upload/51581/51581fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4. Plating of resulting EOC cells suspension. After removing any undissociated tissues, the cell suspension is centrifuged and resuspended in 10 ml of DMEM containing 10% FBS and incubated in a Petri dish at 5% CO2 and 37 °C.
<img alt="Microscope image of leukocytes in urine sample, highlighting cellular morphology for medical analysis." fo:content-width="5in" fo:src="/files/ftp_upload/51581/51581fig5highres.jpg" src="/files/ftp_upload/51581/51581fig5.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5. Morphology of EOC cell cultures immediately after processing. Cell suspension immediately after plating shows EOC as a single cell suspensions and in clumps (both indicated by the arrows), erythrocytes and cell debris still abound at this stage. Original magnification, 20X.
<img alt="Microscope image: cell differentiation with labeled clusters, biological research analysis." fo:content-width="5in" fo:src="/files/ftp_upload/51581/51581fig6highres.jpg" src="/files/ftp_upload/51581/51581fig6.jpg" data-altupdatedat="1747911167000" data-alt="Figure 6"> 
Figure 6. Morphology of EOC cell cultures at day 3 after plating. EOC cell culture at day 3 after plating shows semi-adherent EOC cell cluster (indicated by the arrow) and less erythrocyte contamination. Original magnification, 20X.
<img alt="Figure 7" fo:content-width="5in" fo:src="/files/ftp_upload/51581/51581fig7highres.jpg" src="/files/ftp_upload/51581/51581fig7.jpg"> 
Figure 7. Established EOC cultures after a week from plating. EOC cell culture one week after initial plating shows swirl-like clusters of cells spreading on the tissue culture plastic. Original magnification, 20X.
<img alt="Cell culture microscopy image showing confluency; biological research and analysis tool." fo:content-width="5in" fo:src="/files/ftp_upload/51581/51581fig8highres.jpg" src="/files/ftp_upload/51581/51581fig8.jpg" data-altupdatedat="1747911167000" data-alt="Figure 8"> 
Figure 8. Confluent EOC cultures. Confluent monolayer of EOC cells showing the typical epithelial cobblestone morphology after 14 days in culture.

Watch this Scientific Journal Video about Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens at JoVE.com

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

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Nanyang Technological University

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JoVE Journal

Medicine

13.4K Views.  University of Minnesota.  Deze studie beschrijft een gedetailleerde methode voor de isolatie en karakterisering van primaire eierstokkanker cellen van vaste klinische monsters. Eierstokkanker klinische monsters worden onderworpen aan enzymatische vertering om levensvatbare, fibroblast gratis ovariumcarcinoom (EOC) cellen zeer geschikt voor verdere toepassingen te verkrijgen. 

Video: Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

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.

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 ...

Cancer Research

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

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 ...

Establishing a 3D Ex Vivo Model for Ovarian Cancer-Omentum Interaction

An Ex Vivo Model of Ovarian Cancer Peritoneal Metastasis Using Human Omentum

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian cancer cells as well as immune modulatory signals that allow tumor tolerance. However, we have limited models that closely mimic the interaction between ovarian cancer cells and adipose-rich tissues. To further understand the cellular and molecular mechanisms by which the omentum provides a pro-tumoral microenvironment, we developed a unique 3D ex vivo model of cancer cell-omentum interaction. Using human omentum, we are able to grow ovarian cancer cells within this adipose-rich microenvironment and monitor the factors responsible for tumor growth and immune regulation. In addition to providing a platform for the study of this adipose-rich tumor microenvironment, the model provides an excellent platform for the development and evaluation of novel therapeutic approaches to target metastatic cancer cells in this niche. The proposed model is easy to generate, inexpensive, and applicable to translational investigations.

Author Spotlight: Advanced Ex Vivo Model for Investigating Cancer-Adipose Microenvironment Interaction 

This protocol describes the establishment of a three-dimensional (3D) ex vivo model of cancer cell-omentum interaction. The model provides a platform for elucidating pro-tumor mechanisms within the adipose niche and for testing novel therapies.

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian ...

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 ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...

Modeling Ovarian Cancer Communication for Imaging Mass Spectrometry Analysis: A Method to Identify Small Molecule Chemical Communicators in Metastatic Ovarian Tumor Development

Source: Zink, K. E. et. al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/t/59490">Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry.</a> J. Vis. Exp. (2019)
This protocol describes a technique for sample preparation by co-culturing ovarian explants with the fallopian tube cancer cells. These samples are visualized using imaging mass spectroscopy, or IMS,&#160; to detect small molecule exchange between the cells and tissues leading to tumor development.

Source: Zink, K. E. et. al. Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry. J. Vis. Exp. (2019)
This ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

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