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

During orthotopic transplantation, tumor cells or tissue from a tumor-bearing donor are transplanted into the corresponding tissue of a recipient from the same species. First, prepare a uniform suspension – in a suitable buffer – of fluorescently-labeled brain tumor cells harvested from a tumor-bearing zebrafish. Using a fine gel loading tip, load this cell suspension into a microinjection needle.

Next, secure the loaded needle into the holder of a micromanipulator. Subsequently, transfer an anesthetized zebrafish embryo to a prefabricated injection plate. For easy access during microinjection, position the embryo laterally to visualize the brain ventricles – a network of cerebrospinal fluid-filled cavities within the brain.

Adjust the needle tip to inject the tumor cell suspension into the fourth ventricle, the diamond-shaped cavity of the hindbrain of the embryo. The tumor cells occupy the hollow space within the ventricle. Retract the needle. Transfer the embryo into a culture dish containing saline. Observe the embryo under a fluorescence stereomicroscope to confirm the presence of tumor cells within the ventricle space.

Incubate the embryo. Within hours of injection, the tumor cells migrate and invade the surrounding brain tissue, where they then proliferate giving rise to new tumors. Over time, tumor cells proliferate and invade further, spreading throughout the brain.