Automating Tumor Implantation in Zebrafish Larvae for Cancer Research and Medicine

Treatment personalization is key to improve survival from cancer. As current precision medicine fails to personalize treatment for most patients, how can new functional methods be leveraged to meet this challenge? To facilitate automation of zebrafish to Massimo grafting and related techniques, including hindbrain, subcutaneous, or intravenous drug administration, we provide a thorough protocol for the use of an automated injection system.

This protocol will allow basic researchers and clinical labs to reliably create zebrafish to Massimo graft models, and perform intravenous drug administration to functionally test anti-cancer drug efficacy in vivo. To begin, moisten the agarose gel plate using E3 solution. Hold the plate at an angle to allow any excess E3 solution to pool and collect.

Carefully place 10 to 20 anesthetized larvae onto the moistened plate and allow the excess water to drain away naturally. Then, arrange the larvae so that they're evenly spaced and not touching each other, keeping the minimum distance of 10 millimeters from the plate's edge. Now use a pipette or absorbent paper to remove any excess liquid from the plate while making sure the larvae remain moist.

Next, tap on the Injection Settings menu on the interface. Choose the correct developmental stage of zebrafish larvae suitable for injection, and select the desired injection site from the available options. Then, select the injection location on the schematic diagram of the zebrafish larva.

Adjust the position of the larval diagram, needle tip, and needle orientation to align with the selected injection site. Select the injection macro offering options for automatic and manual modes as preferred. Now, grasp the needle holder firmly and rotate the metal part counterclockwise to detach it from the robot.

Insert the blunt end of a filled needle into the frustoconical end of the needle holder. Tap on Move Stage to mount needle to begin mounting the needle. Once the stage relocates, use the ruler to measure the exposed length of the needle, ensuring it is approximately 22 millimeters.

Then, grasp the needle holder with the filled needle and twist the metal part clockwise to securely tighten the connection. On the pop-up screen, position the needle at the center of the circle and adjust the two screws on the machine. When properly centered, tap the green check mark to confirm.

On the next popup screen, adjust the focus on the needle tip by turning the screw located at the back of the robot head. When the tip is in focus, tap the green check mark. The robot will then automatically calibrate the needle height and rotate the needle while displaying injection positions as red points with each rotation.

When calibration is complete, tap Got It, followed by Continue to Droplet Calibration located at the bottom right of the screen. In the injection interface, click on the Start button to initiate the injection process. The robot stage will begin moving as the system scans the agarose plate to locate a larva.

Once a larva is identified, the needle will automatically move to the selected injection site and adjust its direction to the preset angle. If automatic mode is selected, allow the robot to perform injections seamlessly at the designated site. After each injection, the robot will continue scanning the plate to locate the next larva and proceed with injections.

Once all larvae have been injected, click the Stop button to close the injection interface. The needle will automatically be positioned in the oil well. Then, carefully lift and remove the plate containing the injected larvae.

To transfer the injected larvae, tilt the plate over a Petri dish and use a pipette or a squeeze bottle to gently flush them out with a controlled stream of water. Start from the top of the plate and move downward to flush the larvae effectively. Automated injections into the duct of Cuvier resulted in successful delivery when the injected material was clearly distributed throughout the circulatory system.

However, failed injections resulted in accumulation in the perivitelline space, or only a small number of beads were visible. Injections targeting the perivitelline space were considered successful when fluorescent cancer cells remained localized at the injection site, but were unsuccessful if cells entered circulation were deposited in the yolk or if very few cells were present. Successful hindbrain ventricle injections resulted in microsphere deposition localized to the correct injection site, while errors led to unintended accumulation in the midbrain or forebrain regions.