Name: modeling ovarian cancer metastasis in peritoneal cavity lining: a method to establish an in vitro 3d model of the peritoneal lining
Uploaded: 2023-04-30T13:26:11.000+00:00
Duration: 1 min 44 s
Description: Source: Peters, P. N. et al. Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity. J. Vis. ...

modeling ovarian cancer metastasis in peritoneal cavity lining: a method to establish an in vitro 3d model of the peritoneal lining

Modeling Ovarian Cancer Metastasis in Peritoneal Cavity Lining: A Method To Establish an In Vitro 3D Model of the Peritoneal Lining

To release the normal omentum fibroblasts from culture, rinse the culture flask with 10 milliliters of PBS followed by 3 milliliters of trypsin for no more than 5 minutes. When the cells have detached, neutralize the trypsin with at least three times the volume of full growth medium and transfer the cells to a 50-milliliter conical tube. Spin down the cells and resuspend the pellet in 5 milliliters of full growth medium. Then, count the cells and dilute them to a 2 to 4 times 10 to the third fibroblast per 100 microliters of full growth medium concentration.
Next, add 0.5 micrograms per 100 microliters of rat-tail collagen I to the cells and plate 100 microliters of cells per well into a black, clear bottom 96-well plate. Then, transfer the plate to a cell culture incubator for at least 4 hours or until the cells adhere to the plate surface. While the normal omentum fibroblasts are settling, detach the HPMC from their culture flasks and dilute these cells to 1 to 2 times 10 to the fourth HPMC per 50 microliters of full growth medium.
Then, add 50 microliters of HPMC per well to the attached normal omentum fibroblasts and return the plate to the cell culture incubator for at least 18 hours before experimental use. The next day, seed the 3D organotypic co-cultures with GFP expressing ovarian cancer cells from an 80% to 90% confluent culture at the appropriate dilution for the desired downstream assay.

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Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Cancer Research

Gynecologic Cancer

Assays & techniques

EoE Sub Articles

eoe sub articles

1. Plating the Organotypic Culture
<ol>
	<li>Release NOF (normal omental fibroblasts&#160;) from a 75 cm2 culture flask by rinsing with 10 ml of PBS followed by 3 ml of 0.25% trypsin/25 mM EDTA for no more than 5 min. Neutralize trypsin with at least 3 times the volume of full growth media.</li>
	<li>Transfer trypsinized cells into a 50 ml conical tube and spin down at 0.5 g x 3 min. Remove supernatant and bring cells back up in 5 ml of full growth media. Use a cell counter to count the cells recovered from the culture flask.</li>
	<li>Dilute the cells in the appropriate volume of full growth media to plate 2,000-4,000 NOF per 100 &#181;l onto a black, clear-bottomed, 96-well plate. Add 0.5 &#181;g/100 &#181;l of rat-tail collagen I to the cell mixture before plating. Let the cells sit at 37 &#176;C in 5% CO2&#160;in a humidified environment for at least 4 h, or until the cells adhere to the plate surface.</li>
	<li>Release HPMC (primary human mesothelial cells) from the culture flask using the same methods described in steps 1.1 and 1.2. Dilute the cells in the appropriate amount of full growth media to plate 10,000-20,000 HPMC per 50 &#181;l on top of the NOF already plated with collagen I in 96-well plates. Let the cells sit at 37 &#176;C in 5% CO2&#160;in a humidified environment for at least 18 h before beginning experiments.</li>
	<li>Culture green fluorescent protein (GFP)-expressing HeyA8 ovarian cancer cells in full growth media. HeyA8 ovarian cancer cells are fluorescently labeled by expression of a lentiviral vector expressing copepod cGFP (pCDH-CMV-MCS-EF1). HeyA8 ovarian cancer cells should be 80-90% confluent on the day of use (logarithmic growth phase). Release cells from the culture plate and count using the same methods described in steps 1.1-1.2 for primary cells.</li>
	<li>Allow ovarian cancer cells to recover in full growth media for 15-20 min&#160;at 37&#176;C in a conical tube with the lid loosely sealed.</li>
</ol>

<table><tbody><tr><td>PBS</td><td>Fisher Scientific</td><td>SH3001304</td><td></td></tr><tr><td>15 cm culture dishes</td><td>BD Biosciences</td><td>353025</td><td></td></tr><tr><td>DMEM with L-Glutamine</td><td>Corning</td><td>10-013-CV</td><td></td></tr><tr><td>MEM Vitamins</td><td>Corning</td><td>25-020-Cl</td><td></td></tr><tr><td>MEM Nonessential amino acids</td><td>Corning</td><td>25-025-CI</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Corning</td><td>30-002-CI</td><td></td></tr><tr><td>Shaker</td><td>Thermo-Fisher</td><td>MaxQ 4450</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5702</td><td></td></tr><tr><td>Incubator</td><td>Thermo-Fisher</td><td>Forma Series II Water Jacketed&lt;br/&gt; CO2 Incubator Model 3100</td><td></td></tr><tr><td>Trypsin EDTA, 1x (0.25%)</td><td>Corning</td><td>25-053-CI</td><td></td></tr><tr><td>T-75 Flasks</td><td>BD Biosciences</td><td>353136</td><td></td></tr><tr><td>T-175 Flasks</td><td>BD Biosciences</td><td>353112</td><td></td></tr><tr><td>Pipet tips</td><td>Rainin</td><td>P2, P10, P20, P200 and P1000</td><td></td></tr><tr><td>Pipet tips</td><td>Corning</td><td>Filtered tips P2, P10, P20, P200&lt;br/&gt; and P1000</td><td></td></tr><tr><td>Cell Counter</td><td>Invitrogen</td><td>Countess</td><td></td></tr><tr><td>Countess Cell Counting Chamber&lt;br/&gt; Slides</td><td>Invitrogen</td><td>C10313</td><td></td></tr><tr><td>Trypan Blue Stain (0.4%)</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>Collagen Type I (Rat Tail)</td><td>BD Biosciences</td><td>354236</td><td></td></tr><tr><td>96 well plate, clear bottom, black</td><td>BD Biosciences</td><td>353219</td><td></td></tr></tbody></table>

Source: Peters, P. N. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/t/53541">Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity.</a> J. Vis. Exp. (2015)
In this video, we demonstrate the development of&#160; an in vitro &#160;three-dimensional model of the lining of the peritoneal cavity. This organotypic culture can help to investigate ovarian cancer cell adhesion, invasion, and proliferation.

Source: Peters, P. N. et al. Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity. J. Vis. ...

The peritoneum, or the membrane that lines the abdominal cavity, constitutes peritoneal mesothelial cells supported over a layer of fibrovascular connective tissue. Metastasizing ovarian cancer cells adhere to this peritoneal lining and proliferate into cancerous growths.
To model ovarian cancer metastasis, begin by taking human fibroblasts in a conical tube and mix them with collagen - an extracellular matrix protein. Plate the suspension onto a culture dish. Incubate to facilitate collagen matrix solidification to embed the fibroblasts and mimic the connective tissue of the peritoneum.
Next, add a suspension of mesothelial cells over the fibroblast-collagen layer. Incubate the plate to allow the mesothelial cells to adhere and form a monolayer on the surface of the fibroblast-collagen layer. This 3D organotypic culture now mimics the composition and architecture of an in vivo peritoneal lining.
Finally, seed this organotypic culture with ovarian cancer cells and incubate for the desired duration. The ovarian cancer cells release certain signaling molecules that alter the mesothelial layer, enhancing cancer cell adhesion and invasion, mimicking in vivo metastatic conditions.

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Modeling Ovarian Cancer Metastasis in Peritoneal Cavity Lining: A Method To Establish an In Vitro 3D Model of the Peritoneal Lining

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Modeling Ovarian Cancer Metastasis in Peritoneal Cavity Lining: A Method To Establish an In Vitro 3D Model of the Peritoneal Lining