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