The overall goal of this procedure is to generate primary orthotopic glioma, xenografts to recapitulate, histopathological, and molecular features of malignant glioma subtypes in preclinical animal models. This is accomplished by first obtaining human brain tumor specimens for direct transplantation. The second step is to prepare a cell suspension from the specimen.
Next, the tumor cells are transplanted into the brain of an immunocompromised mouse. The recipient mouse is then monitored for signs of tumor, engraftment and growth, and then harvested for analysis or serial transplantation. Ultimately, histology, DNA sequencing and other molecular assays are used to show infiltrative tumor growth and retention of genetic alterations, such as ISO citrate dehydrogenase mutations.
Preclinical animal models of cancer are essential for investigating tumor biology and therapies. The main advantage of primary glioma xenografts over existing methods such as secondary GLI xenografts, is that glioma cells undergo genotypic and phenotypic changes in culture that cannot be restored when culture glioma cells are transplanted into mice. Demonstrating the procedure.
With me today will be Dr.Gerardo Valez, a member of my laboratory To begin prepare the Pepane Dissociation solutions in a sterile hood with the kit components and instructions supplied by the manufacturer. Place five milliliters of pepane solution in tube one. Tube two should contain three milliliters of the wash protease inhibitor solution, and tubes three through six contain five milliliters.
Each of discontinuous gradient solution. When ready, place the glioma specimen in tube one and tritrate with a five milliliter pipette over a 10 to 20 minute time period. Pipette the material up and down 10 times and then incubate for two to three minutes at room temperature before initiating the next cycle of tation position the tip of the five milliliter pipette closer to the bottom of the conical tube.
With each cycle of tri such that the tip is touching the bottom of the tube for the last two cycles, it is important to avoid the introduction of bubbles during the tation process. The process is completed when there is no or little clearly visible unassociated tissue remaining. After centrifugation, remove the snat and pipette the pellet up and down 10 times in three milliliters of the wash protease inhibitor solution.
Next carefully layer an equal volume of the suspension over each of the tubes containing discontinuous gradient solution. With a pipetter set at the gravity dispense speed, place the tubes in a centrifuge equipped with a swinging bucket rotor. Set the acceleration speed to the lowest setting and turn off the brake before starting the machine.
After retrieving the cells, remove the supernatant resuspend and combine each pellet in five milliliters of sterile balanced salt solution. Place the cells on ice before proceeding. Remove a sample of the cell suspension and determine the density of viable cells by trian blue exclusion with a hemo cytometer.
After an additional centrifugation step, remove the supernatant and resuspend the cell pellet at a density of 25, 000 to 125, 000 viable cells per microliter. Lastly, transfer an adequate volume of the cell suspension to a 200 microliter micro centrifuge tube and place on ice. Begin by confirming the depth of sedation in an anesthetized animal.
Once anesthetized, shave and clean the surgical area with repeated applications of Betadine and ethanol, apply ointment to the eyes. When ready, make a one centimeter midline incision to expose the skull working under a dissecting scope. Identify the suture lines and drill a bur hole through the bone, leaving the dura mater intact.
Prepare the cells for injection by loading the resuspended cells into a Hamilton syringe attached to a probe holder. Introduce the needle into the bur hole so that the beveled portion is just below the skull surface. Advance the needle three millimeters and then withdraw the needle.
0.5 millimeters to create a space for injection. Depress the plunger slowly over 30 seconds, and then allow the needle to remain in the brain for an additional two minutes. Withdraw the needle gradually by ascending 0.5 millimeters and waiting an additional 30 seconds before removing the needle further.
When the injection is complete, remove the mouse from the stereotactic frame and close the wound with an auto clip. Place the mouse on a warming pad and monitor until fully recovered. After euthanasia, carefully remove the brain and place it into a dish containing sterile PBS.
Transfer the brain to a matrix slicer and generate two millimeter coronal slices with razor blades. Next, arrange the coronal slices in a dish containing sterile PBS by this method. The first coronal slice contains the posterior aspect of the olfactory bulbs and the anterior aspect of the frontal lobes.
The engrafted tumor should be most abundant in the right hemisphere of the subsequent three coronal slices, and the injection site is routinely contained in coronal slice. Number three, portions of the tumor may be cryopreserved for future needs with cell viability ranging from 80 to 95%of the five mice transplanted with a glioblastoma. Four demonstrated significant weight loss.
17 weeks after transplantation, Sanger sequencing revealed a heterozygous somatic mutation in the patient. Glioblastoma and primary xenograft immunohistochemistry demonstrated mutant glioma cells densely clustered around blood vessels or in more diffusely infiltrated regions of the patient's specimen and mouse brain. Typical features of an infiltrated glioma with a diffuse tumor mass are seen in the injected right hemisphere.
Invasive growth was found along myelinated tracts of the corpus callosum, the Sato palatal fibers and leptomeningeal dissemination analysis of an anaplastic oligo astrocytoma xenograft after serial passages reveals human cells around blood vessels in stri palatal tracts, and the corpus callosum antibodies. Staining from mutant IDH one protein expression demonstrates that the IDH gene mutation is maintained in a primary orthotopic xenograft following serial passages in recipient mice. To quantify human tumor cells in an associated xenograft for serial passage, human cells are identified in a hemo cytometer under light microscopy by their large size and dark green luminescence.
By flow cytometry, human cells have distinct scatter properties from mouse material and can be gated for further analysis. While attempting this procedure, it's important to be patient primary orthotopic glioma xenografts grow at a significantly slower rate than transplanted cultured cells or cells transplanted into a heterotopic site such as the flank.
