Orthotopic Inoculation of Ovarian Cancer Cells into Murine Ovary: A Procedure to Establish an Ovarian Cancer Mouse Model to Study Peritoneal Dissemination of Cells

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

1. Preparation of Cell Suspensions

1. Human ovarian clear cell carcinoma cell line
   1. Maintain ES-2 cells in RPMI-1640 medium with 10% fetal bovine serum (FBS) and penicillin/streptomycin (penicillin: 100 units/mL, streptomycin: 0.1 mg/mL). Culture cells at 37 °C in a 5% CO₂ humidified incubator.
   2. Collect cells in the logarithmic growth phase in 0.05% trypsin/0.02% EDTA solution (trypsin/EDTA).
      1. First, remove the old media from the cell culture vessel, then wash the cells once with PBS (PBS without Ca²⁺/Mg²⁺,3-4 mL per 25 cm²).
      2. Next, add 1 mL per 25 cm² of trypsin/EDTA. Rotate the cell culture vessel to cover the cell monolayer with trypsin/EDTA. After removing and discarding trypsin/EDTA, return the cell culture vessel to the incubator for 3-5 min or until cells are detached.
      3. Examine the cells under a microscope to ensure the cells are detached from the surface of the cell culture vessel and floating.
      4. Add fresh serum-containing growth medium to inactivate the trypsin (5 mL per 25 cm²) and resuspend the cells. Transfer resuspended cells to a 15 mL conical tube.
      5. Centrifuge at 300 x g for 5 min. Perform centrifugation either at 4 °C or room temperature. Carefully remove supernatant and resuspend cells with growth medium (5 mL per 25 cm²).
   3. Count cells (e.g., by trypan-blue exclusion), then re-suspend in growth medium at a density of 2 x 10⁸ cells/mL. Put the suspended cells on ice.
2. Thaw frozen extracellular matrix (ECM)-based hydrogel aliquots in an ice bath.
3. Add the cell suspension to ECM-based hydrogel aliquots at a ratio of 1:1 (final cell concentration: 1 x 10⁵ cells/µL of medium-ECM-based hydrogel) and mix thoroughly. Place the prepared cell suspensions in an ice bath until use.

2. Orthotopic Inoculation with Cell Suspensions

NOTE: Surgery does not require a dedicated suite. Surgery can be performed on a laboratory benchtop in an area of the room that is separate and has minimal activity. A laminar hood also may be used. Sterile surgical instruments must be used, and scissors should not be used to make skin incisions.

1. Use female nude mice (BALB/c nu/nu) at 8 weeks of age for this orthotopic inoculation model.
2. First, anesthetize the mouse using inhalation with isoflurane (2-4%). Check anesthetic depth by verifying lack of withdrawal upon firm toe pinch. After the mouse is anesthetized, apply a bland sterile ophthalmic ointment to the eyes to prevent drying as necessary.
3. Place the anesthetized mouse on an operating stage, belly-side down. Disinfect the surgical area several times with both alcohol and an iodine-based or chlorhexidine-based scrub.
4. Make a 2-cm incision vertically near the spine between the right costal arch and femur. Use a sterile surgical drape around the incision site. Fix the incised skin surfaces with clamps to maintain the opening.
   NOTE: Retractors can be used instead of clamps.
5. Next, make a second incision (approximately 1 cm) at the parietal peritoneum under the first incision to open the abdominal cavity. Clamp the incised abdominal surfaces. Clip the fat tissue that surrounds the ipsilateral ovary and fallopian tube with a clamp to remove the ovary from the abdominal cavity. Carefully place the ovary and fallopian tube on gauze. Then, place the mouse belly-side down under the dissecting microscope.
6. Place the cell suspension in an insulin syringe (1/2 cc, 29 G) just before injection. Carefully inject the prepared cell suspension (1-2 µL) into the ovary. For several seconds, retain the needle within the ovary to ensure gelation of the ECM-based hydrogel. After gelation, the injected tumor cells remain inside the ovary.
7. Carefully return the ovary into the body. Suture the second incision with sterile medical silk and then seal the skin with wound clips.
8. After surgery, the mice will be administered analgesia (e.g., meloxicam, intramuscular or subcutaneous, 0.2 mg/kg) for initial 24 hr period.