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

Overview

This video demonstrates the orthotopic transplantation of zebrafish brain tumor cells into the fourth ventricle of an immunocompetent zebrafish embryo. This procedure allows for the long-term study of tumor cell invasion and behavior.

Protocol

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.

Materials

Name	Company	Catalog Number	Comments
Egg water	In house		Maintaining embryos, making injection plate
Petri dish	Thermo Fisher	FB0875711Z	Housing embryos, making injection plate
Glass Container	Any commercial brand		Making injection plate
Micropipettes	Any commercial brand		Tumor resuspension and transplantation
Angled Probe	Fine Science Tools	10140-02	Embryo manipulation
Transfer Pipette	Any commercial brand		Embryo manipulation
Stereomicroscope	Olympus	SZ61	Tumor transplantation
Fluorescent Stereomicroscope	Olympus	SZX16	Imaging tumor transplants
Microscope Camera	Olympus	DP-72	Imaging tumor transplants
Incubator	Any commercial brand		Maintaining embryos, warming up injection plate
N2	Any commericial brand		For microinjector set up
Glass capillary (no filament)	World Precision Instruments	TW120-4	Tumor transplantation
Needle puller	Sutter Instruments	P-97	Tumor transplantation
Microloader tips	Eppendorf	930001007	Tumor transplantation
Microinjector	Harvard Apparatus	PLI-90	Tumor transplantation
Tricaine-S (MS-222)	Western Chemical	TRS1	Tumor transplantation, anesthetic
Agarose	Denville	CA3510-8	Making injection plate
Microwave	Any commercial brand		Making injection plate
Scale	Any commercial brand		Making injection plate
Gloves	Any commercial brand		All aspects of the protocol
Pronase	Roche Diagnostics	11459643001	Dechorionate embryos
Razor Blade	Thermo Fisher	12640	Needle preparation
50 mL beaker (2 inch diameter)	Any commercial brand		Making injection plate
Glass Container	Any commercial brand		Making injection plate
Methylene Blue	Sigma-Aldrich	M9140	Add to egg water to prevent fungal growth