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All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. Tissue Processing
<ol>
	<li>Working under sterile conditions, transfer the samples onto a Petri dish (60 mm x 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) (Figures 1A and 1B).</li>
	<li>Transfer the minced tissues into a 15 ml conical 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&#160;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 incubation, transfer (using a 10 ml serological pipette) the cell slurry onto a cell strainer (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 at 4 &#176;C (Figure 2).</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 (Figure 3).</li>
	<li>Change the medium after 24 hr from the initial plating. This allows for removal of cellular debris and the majority of the erythrocytes present in the 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>

No conflicts of interest&#160;declared.

<table><tbody><tr><td>1x PBS, sterile</td><td>Invitrogen</td><td>14190-144</td><td></td></tr><tr><td>Screw lid, polypropylene specimen&lt;br/&gt; container, sterile</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>DMEM medium, sterile</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&lt;br/&gt; blade, sterile</td><td>Fisher Scientific</td><td>forceps: 08-953E, blades:&lt;br/&gt; 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</td><td>Corning</td><td>13-678-11E</td><td></td></tr><tr><td>6 ml Syringe plunger, sterile</td><td>Tyco Healthcare</td><td>8881516911</td><td></td></tr><tr><td>Nylon filter (70 &amp;mu;m ) cell strainer,&lt;br/&gt; sterile</td><td>BD Falcon</td><td>352350</td><td></td></tr><tr><td>5 cm Petri dish, sterile</td><td>Corning</td><td>430166</td><td></td></tr><tr><td>10 cm Petri dish, sterile</td><td>Corning</td><td>430167</td><td></td></tr></tbody></table>

obtaining primary ovarian cancer cells from solid specimens: a method to culture epithelial ovarian cancer cells from ovarian tumor specimens

Source: Pribyl, L. J. et al. <a href="https://www.jove.com/t/51581">Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens.</a> J. Vis. Exp. (2014)
In this video, we describe a method for isolating and characterizing primary ovarian cancer cells from solid ovarian tumor tissue specimens. The epithelial cancer cells obtained using this protocol are suitable for understanding the processes that may lead to ovarian cancer initiation and development.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/20381/20381_Figure1.jpg" /> 
Figure 1. 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="Figure 2" class="xfigimg" src="/files/ftp_upload/20381/20381_Figure2.jpg" /> 
Figure 2. S...

Obtaining Primary Ovarian Cancer Cells from Solid Specimens: A Method to Culture Epithelial Ovarian Cancer Cells from Ovarian Tumor Specimens

Source: Pribyl, L. J. et al. Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens. J. Vis. Exp. (2014)
In this video, we describe a ...

Epithelial cells present on the outer surface of the ovary may undergo malignant transformation to cancerous cells that can grow into ovarian tumors.
To isolate these epithelial ovarian cancer cells or EOCs, begin by taking an ovarian tumor specimen and mince it into small pieces. Transfer the minced pieces into a tube containing a digestion buffer supplemented with the desired protease enzyme.
These proteases degrade the extracellular matrix proteins within the tissue, releasing EOC cells, erythrocytes, and a few dissociated fibroblasts into suspension.
Filter the cell slurry through a strainer to separate the suspended single cells from undissociated tissue pieces. Centrifuge the suspension to pelletize the EOC cells, erythrocytes, and fibroblasts. Discard the enzyme-containing supernatant.
Resuspend the cell pellet into an epithelial growth medium and transfer it into a culture dish. Incubate the culture to allow the EOC cells and fibroblasts to attach to the base of the dish while the erythrocytes float in the media.
Remove the suspended erythrocytes by refreshing the media. Over time, the media facilitates the selective growth of EOC cells over fibroblasts to form a monolayer of primary ovarian cancer cells.

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Research

JoVE Encyclopedia of Experiments

Cancer Research

Gynecologic Cancer

Assays & techniques

1.1K 조회수. 출처: Pribyl, L. J. et al. 고형 표본에서 원발성 난소암 세포를 얻는 방법. J. Vis. 특급. (2014년) 이 비디오에서는 고형 난소 종양 조직 표본에서 원발성 난소암 세포를 분리하고 특성화하는 방법을 설명합니다. 이 프로토콜을 사용하여 얻은 상피암 세포는 난소암의 시작 및 발달로 이어질 수 있는 과정을 이해하는 데 적합합니다. 

비디오: 고체 표본에서 원발성 난소암 세포 채취: 난소 종양 표본에서 상피 난소암 세포를 배양하는 방법 - 개념

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severity1. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible2. Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see Ref. 2). A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers3. To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay4. The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes3,4. This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of &gt;50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies.

Circulating tumor cells are isolated from the blood of cancer patients without inflicting cellular damage. Isolation of tumor cells is accomplished using a bimolecular surface of E-selectin in addition to antibodies against epithelial markers. A nanotube coating specifically promotes cancer cell adhesion resulting in high capture purities.

환자의 혈액 샘플에서 가능한 순환 종양 세포의 신속한 분리

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary ...

연구

JoVE Journal

생체공학

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

인간의 전립선 암 샘플에서 암 줄기 세포의 분리

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

의학

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

인간 세포주에서 Chemoresistant 난소 암 줄기 세포에 대한 심화 학습

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Obtaining Primary Ovarian Cancer Cells from Solid Specimens: A Method to Culture Epithelial Ovarian Cancer Cells from Ovarian Tumor Specimens

내레이션 대본

Obtaining Primary Ovarian Cancer Cells from Solid Specimens: A Method to Culture Epithelial Ovarian Cancer Cells from Ovarian Tumor Specimens