Name: chicken chorioallantoic membrane-based cancer modeling: an in ovo model to study cancer cell tumorigenesis and metastasis
Uploaded: 2023-04-30T13:27:51.000+00:00
Duration: 1 min 41 s
Description: Source: Sharrow, A. C. et al. Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers. J. Vis. ...

chicken chorioallantoic membrane-based cancer modeling: an in ovo model to study cancer cell tumorigenesis and metastasis

Chicken Chorioallantoic Membrane-based Cancer Modeling: An In Ovo Model to Study Cancer Cell Tumorigenesis and Metastasis

To prepare a cancer cell suspension for transplantation, harvest the cells from the cancer cell culture of interest and sediment the cells by centrifugation. Aspirate the supernatant and mechanically resuspend the cells in the residual medium. Place the cell suspension on ice and use a pipette to measure the volume of cells in medium. Then, resuspend the cells at a concentration of 1 to 2 million cells in 20 to 100 microliters of medium, supplemented with 2.7 to 4 milligrams per milliliter extracellular matrix and any growth factors or other additives of interest.
For cancer cell implantation, using a non-stick ring, place up to 6 eggs to be implanted onto the egg rack and roll the edges of the transparent film dressing toward the open window to remove the film from the shells. Check whether the eggs are viable and healthy. Ideal eggs will have a large vessel with smaller branching vessels within the center of the opened area.
Using curved iris forceps, place a sterile non-stick ring onto the CAM over the vessel, ideally over a branch point. Use a sterile glass stir rod to gently abrade the CAM and pipet the cancer cell suspension into the center of the ring. When all of the cells have been delivered, seal the opening with a one quarter piece of 6 by 7 centimeter transparent film dressing and label the egg with an appropriate implant designation. Then, place the egg securely in the incubator, with the opening of the shell facing upright.

chicken-chorioallantoic-membrane-based-cancer-modeling-an-ovo-model

Universidad San Sebastian

Research

JoVE Encyclopedia of Experiments

Cancer Research

Gynecologic Cancer

Animal model studies

EoE Sub Articles

eoe sub articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparing the cancer cell suspension for transplantation (option 1)
NOTE: This is to be completed just prior to the implantation, which should ideally take place between days 7 and 10. Please see notes at the beginning of step 3 or 4 for further information concerning the implantation date. This approach was used for all the cell lines and cultured kidney cancer tumor digests.
<ol>
	<li>Thaw the extracellular matrix solution on ice.</li>
	<li>Using mechanical and/or enzymatic digestion, obtain a single cell suspension using a method appropriate for the cell type being implanted.</li>
	<li>Resuspend the total number of cells to be implanted in an appropriate medium for implantation. Implants of 1-2 x 106&#160;cells per egg are typical. 
	NOTE: The medium used for the implantation of the cell lines is typically the complete medium used for culturing the cells, containing FBS or serum replacement. The implantation of tumor digests typically uses the complete medium used for culturing cell lines of the same cancer type. However, adjustments to the medium formulation may be made if needed experimentally. The effects on tumor development and growth would need to be empirically determined.</li>
	<li>Pellet the cells using a centrifuge speed and time appropriate for the cells being used. Typical speeds are 250-300 x&#160;g, and times are 5-10 min.</li>
	<li>Remove the supernatant from the pelleted cells by pipetting. Mechanically resuspend the cells in the residual medium via flicking or pipetting. Place on ice to cool. Measure the volume of cells and medium using an appropriately sized pipette.</li>
	<li>Calculate implantation volumes such that 1-2 x 106&#160;cells are implanted in a volume of 20-100 &#956;L per egg with a final extracellular matrix concentration of 2.7-4 mg/mL protein. Add medium, any desired growth factors or additives, and extracellular matrix solution according to this calculation. Keep on ice until ready to implant. 
	NOTE: For the calculations in steps 1.3 and 1.6, an extra volume of at least one-half egg implant should be incorporated to ensure an adequate volume for all eggs in the group. For implantation of the ovarian cancer cell lines (i.e., SKOV3 and ID8), 106&#160;cells per egg were implanted. For implantation of the renal cell carcinoma cell line RENCA, cultured cells derived from digested primary human renal cell carcinoma, prostate cancer cell lines (i.e., CWR, C4-2, and MyC-CaP), and bladder cancer cell lines (i.e., HT-1376 and T24), 2 x 106&#160;cells were implanted.</li>
</ol>
2. Preparing tumor pieces for implantation (option 2)
NOTE: This is to be completed just prior to implantation, which should ideally take place between days 7 and 10. Please see notes at the beginning of step 3 or 4 for further information concerning the implantation date. Primary ovarian and bladder cancers were implanted as tumor pieces.
<ol>
	<li>Thaw extracellular matrix solution on ice. Dilute to a final protein concentration of 2.7-4 mg/mL in an appropriate medium containing any desired growth factors or additives. Keep on ice. 
	NOTE: Implantation of tumor pieces typically uses the complete medium used for culturing cell lines of the same cancer type. However, adjustments to the medium formulation may be done if needed experimentally. The effects on tumor engraftment and growth would need to be empirically determined.</li>
	<li>Using a scalpel or scissors, excise pieces from the fresh tumor. Ideal sizes range from 2-5 mm on each side. Keep tissues immersed in the medium until ready to implant.</li>
</ol>
3. Implantation using a nonstick ring (option 1)
NOTE: Cells may be implanted beginning on development day 7 if the CAM is fully developed. Implantation can occur any time prior to hatching that permits adequate time for tumor development and the desired experiment but note that the embryo&#39;s immune cells begin to be present around day 10 postfertilization. Tumor growth rate varies considerably by cell type and needs to be empirically determined for the cell type of interest. The ovarian cancer and the prostate cancer cells were implanted using the nonstick ring method. Note that when a nonstick ring is not available, a pipette tip may be cut to a similar size and used.
<ol>
	<li>Use 70% ethanol to disinfect a biosafety cabinet and all the required tools: an egg rack, curved iris forceps, nonstick rings (1/4 inch inner diameter), glass stir rod, appropriate volume pipettes and tips, 6 x 7 cm transparent film dressing, office scissors, and marker or pencil. Whenever possible, sterile, disposable or autoclaved tools should be used.</li>
	<li>Place eggs to be implanted on an egg rack in a biosafety cabinet. Select a manageable number of eggs. Up to six is typical. Avoid allowing the eggs to cool substantially while working with them.</li>
	<li>Remove transparent film dressing from the shell by rolling the edges towards the open window to avoid pulling away pieces of shell. Check that the eggs are viable and healthy. Ideal eggs will have a large vessel in the center of the opened area with smaller vessels branching from it.</li>
	<li>Using curved iris forceps, place a sterile, nonstick ring onto the CAM over the vessel, ideally over a branch point. Use a sterile glass stir rod to gently abrade the CAM.</li>
	<li>Pipet the cell suspension from step 1.6 into the center of the nonstick ring. Alternatively, use forceps to place a tumor piece from step 2.2 into the center of the nonstick ring and cover with 20-50 &#956;L of the extracellular matrix solution generated in step 2.1.</li>
	<li>Seal the opening with a one-quarter piece of 6 x 7 cm transparent film dressing. Label the eggs with an appropriate implant designation. 
	NOTE: Numbering the eggs within a group facilitates longitudinal observations.</li>
	<li>Return the egg to the incubator. Ensure that the opening of the shell sits upright and that the egg is secure. 
	NOTE: Eggs can be returned to an egg incubator without rotation. Alternatively, high postimplant viability can be obtained using a 37-38 &#176;C cell incubator with the CO2&#160;deactivated and a hygrometer to monitor the humidity, which should be 50%-80%.</li>
</ol>

<table><tbody><tr><td>010 Teflon (PTFE) White 55 Duro Shore D O-Rings</td><td>The O-Ring Store</td><td>TEF010</td><td>Nonstick ring for cell seeding. 1/4&quot;ID X 3/8&quot;OD X 1/16&quot;CS Polytetrafluoroethylene (PTFE).</td></tr><tr><td>Delicate Operating Scissors; Curved; Sharp-Sharp; 30mm Blade Length; 4-3/4 in. Overall Length</td><td>Roboz Surgical</td><td>RS6703</td><td>This is provided as an example. Any similar curved scissors would work as well.</td></tr><tr><td>Dremel 8050-N/18 Micro 8V Max Tool Kit</td><td>Dremel&amp;nbsp;</td><td>8050-N/18</td><td>This kit contains all necessary tools</td></tr><tr><td>Fertilized chicken eggs (Rhode Island Red - Brown, Lab Grade)</td><td>AA Lab Eggs Inc.</td><td>N/A</td><td>A local egg supplier would need to be identified, as this supplier only delivers regionally.</td></tr><tr><td>Hovabator Genesis 1588 Deluxe Egg Incubator Combo Kit</td><td>Incubator Warehouse</td><td>HB1588D-NONE-1102-1588-1357</td><td>Other egg incubators may be used, but their reliability would need to be verified. After implantation, a cell incubator with the CO2 disabled may also be used.</td></tr><tr><td>Incu-Bright Cool Light Egg Candler</td><td>Incubator Warehouse</td><td>1102</td><td>Other candlers may be used; however, this is preferred among those that we have tested. This candler is included in the aforementioned incubator kit.</td></tr><tr><td>Matrigel Membrane Matrix HC; LDEV-Free</td><td>Corning</td><td>354248</td><td>Extracellular matrix solution</td></tr><tr><td>SKOV3</td><td>ATCC</td><td>HTB-77</td><td>Human ovarian cancer cell line.</td></tr><tr><td>Stirring Rods with Rubber Policeman; 5mm diameter, 6 in. length</td><td>United Scientific Supplies</td><td>GRPL06</td><td>This is provided as an example. Any similar glass stir rods would work as well.</td></tr><tr><td>Specimen forceps</td><td>Electron Microscopy Sciences</td><td>72914</td><td>This is provided as an example. The forceps used for pulling away the shell for bioluminescence imaging are approximately 12.8 cm long with 3 mm-wide tips.</td></tr></tbody></table>

Source: Sharrow, A. C. et al. <a href="https://www.jove.com/t/60651">Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers.</a> J. Vis. Exp.&#160;(2020)
In this video, we describe the implantation of cancer cells onto a chicken chorioallantoic membrane to generate an in ovo model. The model successfully engrafts the ovarian cancer cells to study the progression of gynecological cancer.

Source: Sharrow, A. C. et al. Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers. J. Vis. ...

The chicken chorioallantoic membrane, or CAM, is a highly vascularized membrane found in the eggs of certain vertebrates like chickens. This extra-embryonic membrane lines a non-vascular shell membrane and functions as the site of exchange of gaseous oxygen and carbon dioxide between the embryo and the environment.
To generate a CAM cancer model, begin with a viable, fertilized chicken egg bearing a fully developed CAM with a partially opened shell that exposes its branching vasculature. Confirm that the CAM is detached from the shell membrane.
Place a sterile non-stick ring over a blood vessel branch point. Using a glass rod, gently rub the membrane inside the ring to scrape its outer epithelial layer and expose the underlying mesodermal layer carrying the vasculature. Pipet a chilled suspension of cancer cells mixed with a suitable extracellular matrix into the ring. The ring restricts cellular dispersal.
Allow the matrix to solidify to secure the cells within. Seal the eggshell opening and incubate the egg under appropriate conditions. The dense vasculature within the mesodermal layer supplies oxygen and essential nutrients to the cancer cells, thus providing a suitable microenvironment for their growth and expansion.

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Chicken Chorioallantoic Membrane-based Cancer Modeling: An In Ovo Model to Study Cancer Cell Tumorigenesis and Metastasis

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Chicken Chorioallantoic Membrane-based Cancer Modeling: An In Ovo Model to Study Cancer Cell Tumorigenesis and Metastasis