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

Published: April 30, 2023

Abstract

Source: Casey, M. J. et al. Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response. J. Vis. Exp. (2017)

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.

Disclosures

The authors have nothing to disclose.

Materials

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

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Cite This Article
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. J. Vis. Exp. (Pending Publication), e20689, doi: (2023).

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