Overview
This video describes the protocol for creating a murine experimental model to study tumor development and peritoneal metastasis. This ovarian cancer model is generated by the orthotopic inoculation of human epithelial ovarian cancer cells into the ovaries of a female mouse.
Protocol
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
- Human ovarian clear cell carcinoma cell line
- 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.
- Collect cells in the logarithmic growth phase in 0.05% trypsin/0.02% EDTA solution (trypsin/EDTA).
- 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).
- 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.
- Examine the cells under a microscope to ensure the cells are detached from the surface of the cell culture vessel and floating.
- 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.
- 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).
- 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.
- Thaw frozen extracellular matrix (ECM)-based hydrogel aliquots in an ice bath.
- 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.
- Use female nude mice (BALB/c nu/nu) at 8 weeks of age for this orthotopic inoculation model.
- 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.
- 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.
- 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. - 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.
- 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.
- Carefully return the ovary into the body. Suture the second incision with sterile medical silk and then seal the skin with wound clips.
- After surgery, the mice will be administered analgesia (e.g., meloxicam, intramuscular or subcutaneous, 0.2 mg/kg) for initial 24 hr period.
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Materials
Name | Company | Catalog Number | Comments |
Matrigel matrix | BD | 354234 | ECM-based hydrogel |
Insulin syringe | TERUMO | SS-05M2913 | 1/2 cc, 29 G x 1/2" |
Suturing needle with suture | 11 mm, 3/8, Nylon6-0 | ||
Reflex 7 mm Wound Clip Applier | CellPoint Scientific | 204-1000 | |
Reflex 7 mm wound clips | CellPoint Scientific | 203-1000 |