Long-term Orthotopic Transplantation of Tumor Cells into Zebrafish Embryos: An Efficient Method to Deliver Tumor Cells into the Fourth Ventricle of Zebrafish Embryos for Long-term Analysis of Tumor Cell Behavior

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

1. Injecting the Tumor Suspension into the Fourth Ventricle of a 2-dpf Embryo

1. Transfer 10–20 anesthetized dechorionated embryos to the periphery of the injection plate using a transfer pipette; the embryos should fall laterally onto the injection plate, with the ventricles clearly visible and accessible. Use an angled probe to adjust the embryos as needed and position them away from the outer edge of the injection plate.
2. Load 1–2 µL of the tumor cell suspension into the injection needle using gel loading pipette tips and insert the needle into the manipulator.
3. Manually lower the manipulator, holding the needle at a 45° angle. Adjust the knobs on the micromanipulator in the x, y, and z directions until the needle is just above and approximately 5 mm to the right of the embryo head. Look through the eyepieces of the stereomicroscope and slowly adjust the micromanipulator in the x-direction until the needle pierces the fourth ventricle of the embryo. Do not allow the needle to pierce the heart or the yolk.
   1. Push the foot pedal attached to the microinjector to inject the tumor cell suspension.
      NOTE: The injection pressure and time will vary based on needle bore size, but a good starting point is 5–10 PSI and 50–100 ms. The volume of injected tumor cells can be assessed with standard oil drop techniques through injection into mineral oil, if desired. Injections of 500 pL with a drop diameter of 0.1 mm typically give the best results.
4. Gently rinse the injected embryos off the injection plate and into a Petri dish using fresh egg water (0.6 g/L aquarium salt in reverse osmosis water + 0.01 mg/L methylene blue). Maintain the embryos in egg water for the remaining procedures or until they are grown in tanks.
5. Inspect the injected embryos under a fluorescence stereomicroscope. Confirm that the injection pressure, angle, needle size, and cell suspension viscosity result in tumor cells filling 25–50% of the ventricle space. Adjust these parameters as necessary.
6. Repeat steps 1.1–1.5 for any remaining embryos. Anesthetize the additional embryos as needed.    
   NOTE: An experienced user can inject up to 300 embryos over 3–4 h.
7. Place the embryos back in the 28°C incubator overnight.
8. The next day, assess embryo survival by examining morphological and physiological features, such as normal heart and brain development, as described.