The overall goal of this procedure is to author topically allograft medulloblastoma tumor cells into an immunocompromised host to initiate a secondary tumor. This is accomplished by first associating primary medulloblastoma tissue into single cells and small cellular aggregates. The second step of the procedure is to prepare immunocompromised mice for intracranial injection.
The third step of the procedure is performing microinjection of tumor cells into the host cerebellum. The final steps of the procedure are maintaining and observing the tumor cell grafted hosts. Ultimately, results can be obtained that show secondary medulloblastoma formation in the hosts by the displayed neurological symptoms and histological analysis.
This technique can help answer key questions in cancer biology, such as determining the cellular origin of a tumor and factors important for initiating and propagating tumors, Microdisect and dissociate tumor tissue from m sparing medulloblastoma as described in the companion video article, isolation, enrichment and maintenance of medulloblastoma cell lines dilute the tumor cell suspension in neuro stem cell medium, so that four microliters of solution contains the appropriate number of dissociated tumor cells. Here, five times 10 to the six cells. Keep the dilute cell suspension in a 200 microliter micro centrifuge tube on ice.
Use a hair clipper to shave the dorsal posterior head region of an anesthetized immunocompromised host mouse revealing the posterior skull. Apply Betadine solution to the exposed scalp, followed by wiping with an alcohol pad. Repeat these two sterilizing steps twice.
Use a sterile scalpel to make a one-quarter inch incision in the posterior scalp. Use a sterile dental drill to drill a 0.5 millimeter bur hole, two millimeters to the right and two millimeters posterior to lambda. Hook the host mouses in sizes onto a stereotactic frame.
To secure the mouse load a bevel Tip 10 microliter syringe. With four microliters of tumor cell suspension, secure the syringe to a micro manipulator on the frame. Under the microscope, carefully descend and position the needle tip into the bur hole.
When the bevel of the syringe is below the skull surface, descend for three millimeters. Then back up the 0.5 millimeters to create a space in between the tip of the needle and the cerebellar tissue. Slowly with steady force over the course, 30 seconds, inject four microliters of the tumor cell suspension into the cerebellum of the host mouse.
Leave the needle in place for two minutes. Remove the needle and the syringe. Use a sterile auto clip to seal the wound.
Allow the mouse to recover on a warming blanket. After surgery, place the mouse back in a sterile housing cage and monitor behavior and for infection at the injection site. Seven days after surgery, remove the auto clip.
Mice will display typical medulloblastoma symptoms, symptoms between two and six months. Here is a typical example of secondary medulloblastoma tissue developed 26 days after injection of five times 10 to the five primary tumor cells. Hemat toin staining reveals the typical cellular morphology of medulloblastoma, including high nuclear to cytoplasmic ratio and nuclear polymorphism.
These secondary tumor cells are highly proliferative, as indicated by KI 67 expression, and they also robustly express membranous smoothened M two YFP. Using the companion protocol for primary mouse modelo blasts, stoma cells. These secondary modelo blasts stoma cells can be cultured and expanded for multiple passages.
Red arrows point to some red blood cells. Here is the same Representative tumor colony cultured at four days and at 14 days. The cultured tumor cells are bipolar, elongated with scan cytoplasm, and often radiate from a dense core of cellular aggregates.
While attempting these procedures, it's important to create a proper space in between the cere tissue and the tip of injection needle by sending it 0.5 millimeters after lowering it three millimeters beneath the skull surface.
