The overall goal of this procedure is to demonstrate surgical placement of human anaplastic, thyroid carcinoma, or a TC cells into the thyroid in order to establish an orthotopic xenograft mouse model. This is accomplished by first culturing and harvesting a TC cells. After preparing the mouse for surgery, expose the thyroid by two successive longitudinal incisions in the neck and inject the A TC cells slowly into the right thyroid gland.
The final step is to close the incisions with sutures and to allow the mouse to recover. Ultimately histological examination of specimens from resected tissues are used to show tumor growth and invasion under a variety of conditions. The main advantage of this technique over existing methods such as subcutaneous xenograft transplant, is that orthotopic transplantation places a TC cells in their biological niche and closely replicates disease progression and pathology as it occurs in humans.
This method can help answer key questions such as determining the biological mechanisms of thyroid cancer aggressiveness. Begin this procedure by culturing the human A TC cell line TJ 11 T in six Well culture plates using complete RPMI 1640 supplemented media one day prior to cell harvest when the cells are 70 to 80%confluent thaw, 0.01 milliliters of major gel at four degrees Celsius. Harvest the cells using trypsin once they have become 80 to 90%confluent once the cells have been pelleted, aspirate the media and resuspend cells in two milliliters of supplemented RPMI 1640 Media for each six well plate harvested.
Next, determine the cell density by mixing the cell suspension one to one with 0.4%Triam blue dye determine cell density using a TC 10 automated cell counter. Calculate the volume of the cell suspension needed to inject 500, 000 cells per mouse. Ensure adequate cells are available for the implantation by calculating for double the number of planned implants.
Transfer the required volume of cell suspension into a 15 milliliter conical tube. Next centrifuge at 200 Gs for four minutes at room temperature. Then aspirate the media and resuspend cells at 100 million cells per milliliter in complete RPMI 1640 supplemented media.
Next, transfer cells to a 1.6 milliliter tube. Add one volume of matri gel and mix gently by slowly pipetting in and out. Place the cell suspension on ice until it is needed.
First, sterilize the surgical zone, which includes the dissecting scope and surrounding area by wiping all surfaces with 70%ethanol. Then prepare the recovery area by turning on heating lamps and putting down sterile pads. Anesthetize the mouse and prepare it for surgery by first shaving the neck region of the mouse from jawline to top of sternum and out to each arm.
Next, screw up the surgical area three times by alternating chlorhexidine soaked gau squares and ethanol. Then one final time with Betadine soaked gauze. Also gently apply eye ointment to prevent eyes from drying.
At this point, check the pedal reflex to ensure that the mouse is adequately sedated. Then place the mouse dorsal recumbent on a disposable sterile field barrier. Under the dissecting scope, secure the mouse in place with cloth tape.
Next, thoroughly scrub hands and fingernails and put on sterile surgical gloves in a sterile fashion, open surgery pack and arrange instruments. Finally, place a sterile drape over the mouse, leaving only the surgical site exposed. Begin by making a one to 1.5 centimeter longitudinal incision along the midline of the throat using a sterile disposable scalpel.
Do not deviate from the midline as this would increase the chance of nicking or severing large arteries. Make a second incision into the strap muscles surrounding the trachea. Then pull the right side of the incised muscle to the side, exposing the right thyroid gland.
Have an assistant hand over the syringe. Slowly inject it into the right thyroid gland using a 31 gauge insulin syringe. Then gently remove the needle suture together the muscle layer with six oh nylon monofilament suturing material using an interrupted suturing style.
Finally, stitch together the skin in the same fashion postoperatively. Apply a layer of triple antibiotic ointment directly over the incision site. Allow the mouse to recover dorsal recumbent under a lamp.
Once the mouse can achieve sternal recumbent without assistance, it can be placed back with other mice in its cage. Check the incision site and overall health of the mouse daily for a week following surgery. Then check it once a week.
If at any time the mouse exhibits signs of declining health, it should be euthanized and its tissues collected. Once mice have reached a predetermined postoperative time point anesthetize with ketamine and xylazine and perfuse the animal tardily with buffer, followed by fixative, then harvest the tissues and process them for histology. The thyroid and trachea from a non injected control animal are shown here with hematin and eosin staining follicles display a close yet loose association and have essentially round morphology.
In contrast, mice receiving an orthotopic injection of 500, 000 A TC cells exhibit significant infiltration of cancer cells into the thyroid by four weeks post injection. The nature of the invading cells shows characteristic spindle shaped cells and medium to giant size cells with eosinophilic cytoplasm and large nuclei. Due to the small size of the thyroid and the use of injectable delivery, unintentional outcomes may arise such as the development of a tumor mass outside of, but not encompassing the thyroid.
This is due to needle penetration through the thyroid when cells are expelled Occasionally, mice presenting with no tumor growth, both grossly and histologically are detected as shown here. Absence of detectable tumor development is caused by expulsion of the cell suspension from the injection site into neighboring tissues and cavities Once mastered, this technique can be performed within 20 minutes. After watching this video, you should have a good understanding of how to generate your own orthotopic model for anaplastic thyroid carcinoma.
By surgically accessing the thyroid gland and injecting cultured a C cells.
