orthotopic inoculation of thyroid cancer cells into murine thyroid: a procedure to establish orthotopic thyroid cancer mouse model

Orthotopic Inoculation of Thyroid Cancer Cells into Murine Thyroid: A Procedure to Establish Orthotopic Thyroid Cancer Mouse Model

For orthotopic thyroid tumor cell inoculation, suspend an aliquot of Cal62 cells in 5 microliters of PBS, and subcutaneously inject 100 microliters of analgesic and 100 microliters of antibiotic into a 7-week-old BALB/c nude mouse. After confirming a lack of response to toe pinch, place the animal under a dissecting microscope and disinfect the neck of the animal with iodopovidone.
Next, make an approximately 2-centimeter incision in the skin and displace the salivary glands to expose the neck. Use dissection forceps and/or scissors to dissect the strap muscles to expose the trachea and thyroid gland, and use a 10-microliter syringe to inject the 5-microliter volume of tumor cells into the right thyroid lobule, located at the side of the cricoid cartilage.
When all of the cells have been delivered, reposition the salivary glands and use silk-braided, coated, non-absorbable sutures to close the incision. Then, apply iodopovidone to the wound area and place the mouse on a thermic blanket with monitoring until full recovery.

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Head and Neck Cancer

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Thyroid Orthotopic Inoculation of Cells 
<ol>
	<li>Cell preparation
	<ol>
		<li>Engineer a Cal62 human thyroid cancer cell line to overexpress a transgenic construct that constitutively expresses GFP and luciferase. Select the transgenic cells by antibiotic resistance or by sorting the GFP-positive cells with flow cytometry.</li>
		<li>Grow these cells in DMEM supplemented with antibiotics (penicillin and streptomycin) and 10% FBS at 37 &#176;C and 5% CO2.</li>
		<li>Suspend 1 x 105&#160;cells in 5 &#956;L of PBS.</li>
	</ol>
	</li>
	<li>Cell inoculation into the thyroid gland of mice
	<ol>
		<li>Inject 100 &#956;L of analgesic (buprenorphine) and 100 &#956;L of antibiotic (cephalosporin) subcutaneously into 7-week-old BALB/c&#160;nu/nu&#160;mice.&#160;Disinfect the injection site with alcohol.</li>
		<li>Inject 5 &#956;L of the cell solution into the thyroid gland of the mice.
		<ol>
			<li>Anesthetize the mouse using 3% isoflurane mixed with oxygen and place it under a stereomicroscope in a sterile flow cabinet.&#160;&#160;Assess the level of anesthesia by pedal reflex (firm toe pinch) and adjust anesthetic delivery as appropriate to maintain the surgical plane.</li>
			<li>Apply ophthalmic ointment to both eyes to prevent desiccation.&#160;</li>
			<li>For each mouse, disinfect the neck with iodopovidone and make an incision of approximately 2 cm in the skin with scissors. Once open, expose the neck by displacing the salivary glands.</li>
			<li>Dissect the strap muscles using dissection forceps and/or scissors to expose the trachea and the thyroid gland. Using a 10 &#956;L syringe, inject 5 &#956;L of the cell solution into the right thyroid lobule, located at the side of the cricoid cartilage.</li>
			<li>Reposition the salivary glands and suture the incision with silk using braided, coated, non-absorbable sutures.</li>
			<li>Add iodopovidone to the wound area and place the mouse on a thermic blanket while it recovers from the anesthesia.</li>
		</ol>
		</li>
		<li>The following day post-surgery, inject subcutaneously analgesic (buprenorphine) and add 3 mL of liquid ibuprofen (40 mg/mL) into 250 mL of the drinking water for 1 week.</li>
	</ol>
	</li>
</ol>

<table><tbody><tr><td>Basement Membrane Matrix: Matrigel Basement Membrane Matrix High Concentration</td><td>Corning</td><td>#354248</td><td></td></tr><tr><td>In vivo&amp;nbsp;delivery reagent: Invivofectamine 3.0 Reagent</td><td>Thermo Fisher</td><td>IVF3005</td><td></td></tr><tr><td>In vivo&amp;nbsp;imaging software: IVIS-Lumina II Imaging System</td><td>Caliper Life Sciences</td><td></td><td></td></tr></tbody></table>

Source: Julia Ramirez-Moya, et al. <a href="https://www.jove.com/video/59322" target="_blank">In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice.</a> J. Vis. Exp. (2019)
This video describes the procedure for orthotopic inoculation of thyroid cancer cells into the thyroid gland of mouse models, which may then serve as a valuable platform to assess carcinogenesis and discover potential treatment strategies.

Source: Julia Ramirez-Moya, et al. In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice. J. Vis. Exp. (2019)
This video describes the procedure ...

Orthotopic inoculation facilitates the delivery of cancer cells to a recipient animal at an organ or site that matches the tissue histotype. To begin, prep an anesthetized immunocompromised nude mouse in the supine position.
Next, make a surgical incision in the neck region to reveal the salivary glands. Displace the salivary glands and dissect the strap muscles to expose the underlying trachea and thyroid gland.
The thyroid gland, an endocrine gland, consists of two lobes that lie on either side of the trachea and surround the cricoid cartilage. These two lobes are connected by a thin strip of thyroid tissue called the isthmus.
Now, prepare a suspension of thyroid cancer cells with a suitable buffer in a syringe. Carefully inject the cell suspension into the mouse&#8217;s thyroid lobe.
Subsequently, reposition the salivary glands and suture the incision. Allow the mouse to recover. Monitor the mouse weekly to observe the growth of orthotopic tumors within the thyroid lobe.

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Orthotopic Inoculation of Thyroid Cancer Cells into Murine Thyroid: A Procedure to Establish Orthotopic Thyroid Cancer Mouse Model

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Orthotopic Inoculation of Thyroid Cancer Cells into Murine Thyroid: A Procedure to Establish Orthotopic Thyroid Cancer Mouse Model