Intra-uterine Cancer Cell Injection: A Technique to Deliver Cancer Cells for Establishing Intra-peritoneal Ovarian Cancer Mouse Model

Overview

In this video, we demonstrate intra-uterine injection of ovarian cancer cells in a mouse model. This orthotopic murine model helps us to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention and treatment of recurrent ovarian cancer.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Intra-uterine Injection of Human Ovarian Cancer Cells in Athymic Nude Mice

NOTE: The following procedure requires assistance from a second person. In addition, since this is survival surgery, use sterile surgical instruments. This survival surgery procedure should take approximately 10 to 15 min.

1. Anesthetize the mouse using 2% isoflurane. 7- to 8-week-old athymic nude mice are typically used.
2. Verify that the animal is completely anesthetized by pinching the footpad using fingers or forceps. The animal should be completely non-responsive to the pain before starting the surgery.
3. Place the anesthetized mouse with its right lateral side up, on a sterile gauze pad with the head away from the investigator.
4. Apply ointment on the eyes with a cotton-tip applicator to prevent dryness while under anesthesia.
5. Immediately insert the head in a nose cone system connected to an isoflurane vaporizer (Fig. 1A). This delivers the anesthesia throughout the surgical procedure.
6. Disinfect the left (or right) side of the abdomen using alcohol pads (Fig. 1B) followed by iodine, then place a sterile surgical drape over the surgical site.
7. Using sterile surgical scissors and forceps, make a 1-2 cm skin incision on the lower left quadrant of the mouse (Fig. 1C).
8. Lift the muscle and make an incision to reach the peritoneum.
9. Dissect the oblique muscle to expose the abdominal cavity.
10. Locate and identify the left uterine horn (Fig. 1D).
11. Using a sterile hemostat, clamp both anterior and posterior sides of the horn (Fig. 1E). Place the anterior clamp right below the fallopian tube and the posterior clamp right above the cervix.
12. Have the second person inject the cell suspension. Place the needle containing cells at a 45° angle perpendicular to the horn (Fig. 1F). Very slowly inject 50 µl of cell suspension into the lumen of the uterine horn.
13. Remove the needle and release the anterior clamp.
14. Release the posterior clamp and replace the uterine horn in the abdominal cavity.
15. Close the peritoneum using a synthetic absorbable suture and close the skin using a tissue adhesive.
16. Remove the mouse from the nose cone and place it back in the cage. If necessary, change the beddings to ensure a clean cage after the surgery. Make sure all animals are awake and active before leaving unattended.
17. Provide Ibuprofen at 0.11 mg/ml of drinking water for the first 48 hr post-surgery. Afterward, replace with regular drinking water.
    NOTE: that if athymic nude mice are used, and the skin is closed using tissue adhesive instead of sutures, it is not necessary to separate the mice after surgery. SCID mice are more aggressive and may have to be separated.
18. Closely monitor the site of incision for possible infection due to open wound.

Representative Results

Figure 1: Intra-uterine injection of ovarian cancer cells. (A) anesthesia is continuously administered via a nose cone; (B-E) skin is incised to locate and clamp the uterine horn; (F) cancer cells are injected slowly into the lumen of the uterus.

Materials

Name	Company	Catalog Number	Comments
Alcohol pads	Fischer Scientific	06-669-62
Athymic nude mice	Harlan
Hemostat	VWR	82027-422
Ibuprofen	Walgreens		Children's Ibuprofen 100 (100 mg/5 ml)
Isoflurane	Butler Schein	NDC 11695-6776-1
Surgical forceps	VWR	82027-386
Surgical scissors	VWR	82027-584
Synthetic absorbable suture	Covidien	SL-636
Tissue adhesive	Vetbond	1469SB
1 ml syringe	Becton Dickinson	309602
25 gauge needle	Becton Dickinson	305122
Paclitaxel	Hospira, Inc.	NDC 61703-345-50
Puralube	Pharmaderm