Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

The overall goal of the following experiment is to generate genetically engineered astrocytoma models from defined cell types for phenotypic characterization in vitro and in vivo. This is achieved by harvesting wild type primary cells from non Cree expressing neonatal conditional genetically engineered mice for in vitro culture. In the second step, the primary cells are infected with an adenoviral vector encoding Cree recombination to induce genetic recombination of the F flox alleles in vitro.

Next, the recombined cells are serially cultured for their phenotypic characterization. Ultimately, whether the defined mutations promote glio agenesis in specific neural cell types is determined by characterization of tumorogenic relevant phenotypes in vitro and in vivo. This method can help answer key neuro-oncology questions such as What are the phenotypic consequences of astrocytoma associated mutations?

The implications of this technique extend to preclinical drug development for astrocytomas, because these genetically defined cells can be used in vitro and in vivo, in immune competent syngenetic mice. Visual demonstration of this method is critical as the cell harvest and orthotopic injection steps are technically difficult and require accurate identification of anatomical structures. Demonstrating the procedure will be Ryan Bash, a technician from my laboratory To harvest the brain tissue.

Begin by using ethanol sterilized dissecting scissors to make a sagittal cut in the skin over the cranium of a one to three day old euthanized neonatal, genetically engineered mouse from the spinal cord to the nose. Next, make a cut in the cranium along the sagittal suture, starting from the spinal cord and extending past the olfactory bulbs. Then use curved forceps to gently peel each hemisphere of the cranium laterally away from the brain using straight micro forceps.

Next, gently pinch away the dorsal portion of each cortical hemisphere taking care to avoid the cerebellum and olfactory bulb and place the brain tissue into a tissue culture dish containing H-B-S-S-N. Under a dissecting microscope, use two pairs of micro forceps to gently remove the meninges from each cortical hemisphere and place the tissue culture dish back onto the ice to homogenize the brain tissue. First, use a clean razor blade to finally dice the cortical hemispheres.

Then move the plate to a tissue culture hood and transfer the tissue pieces into a sterile 15 milliliter conical tube. Rinse the plate with one milliliter of ice cold HBSS and transfer the wash to the tube as well. After removing the excess HBSS at two milliliters of room temperature tripsin with EDTA to the tube and associate the tissue fragments further by pipetting up and down about 10 times with a one milliliter pipette.

Incubate the cell suspension at 37 degrees Celsius for 15 to 20 minutes. Carefully mixing the cell solution by inversion every five minutes after the incubation, mixed three milliliters of complete media into the cell suspension to inhibit the trypsin by pipetting up and down 10 times as just demonstrated. Then spin down the dissociated astrocytes.

Carefully remove the snat with a one milliliter pipette and resuspend the pellet in four milliliters of 37 degrees Celsius. Complete media. Then transfer the astrocyte suspension to a T 75 screw top tissue culture flask, and maintain the cortical astrocytes at 37 degrees Celsius in 5%carbon dioxide approximately 16 hours after the harvest.

Wash the flask with four milliliters of 37 degrees Celsius HBSS. Then add four milliliters of complete media and maintain the cortical astrocytes at 37 degrees Celsius in 5%carbon dioxide. When the culture reaches about 95%confluence, shake the culture overnight at 37 degrees Celsius in 5%carbon dioxide.

Then remove the media containing the detached cells and wash the plate with four more milliliters of 37 degrees Celsius HBSS. Then add four milliliters of fresh complete media and incubate the cells again 24 hours after enriching the culture for cortical astrocytes. Refresh the culture with two milliliters of complete media.

Then incubate the cells with one milliliter of complete media containing one microliter of at least one times 10th of the 10th pfu per milliliter of the adenovirus of interest at 32 degrees Celsius in 5%carbon dioxide at five. Gfab CRE infection causes a proliferative advantage in recombined gfab astrocytes increasing the astrocyte purity from 59%to greater than 90%after five to nine passages in culture. After six hours, remove the virus containing media and re incubate the culture in fresh, complete media.

After the cortical astrocytes are harvested at about 90%confluence and the appropriate cell number are resuspended in ice cold 5%Methylcellulose. Place a 250 microliter glass syringe into a repeating antigen dispenser, and then attach a blunt 18 gauge needle to the syringe. Use the 18 gauge needle to aspirate the cortical astrocytes into the syringe and remove any air bubbles.

Then discard the 18 gauge needle and fit a 27 gauge needle onto the syringe. Press the button on the antigen dispenser until fluid is expelled from the new needle to implant the astrocytes. Next, secure a three to six month old immunocompetent recipient animal within a stereotaxic frame and make an incision over the sagittal suture approximately 0.5 centimeters long between the ears and eyes.

Locate the intersection of the coronal and sagittal sutures or the BRE MA, as well as the intersection of the lambdoid in sagittal sutures or the Lambda. After ensuring that BMA and lambda are in the same horizontal plane, attach the syringe and the repeating antigen dispenser to the stereotaxic frame over the animal's head, and to bring the tip of the needle into contact with the bgma. Raise the needle slightly off the skull surface and move two millimeters lateral and one millimeter rostral from the bgma.

Then lower the needle carefully through the skull to its destination. Four millimeters ventral from the breg MA and activate the repeating antigen dispenser. One time to inject five microliters of the cell suspension.

Leave the needle in place for two minutes to allow the intracranial pressure to equilibrate and then withdraw the needle slowly over a period of 30 seconds using a cotton swap to apply pressure to any bleeding that may occur. Finally, use tissue adhesive to approximate the wound edges and to close the incision, and then place the animal in a clean, warm cage to recover. Xenografts of established human cell lines require immunodeficient hosts and generally to not recapitulate the histopathology of human astrocytomas.

For example, U 87 MG orthotopic xenografts form circumscribed tumors that do not invade a normal brain. In contrast, injection of T RRP null astrocytes into the brains of immune competent syngeneic mice yields tumors that recapitulate the histological features of their human counterparts, particularly the invasion of normal brain tissue. To monitor the TRP null allograft growth, mice were sacrificed every five days after cell injection, and the tumor burden was determined by quantifying the tumor area on h and e stained brain sections.

The tumor area was determined to increase exponentially over time, and the orthotopic injection of one times 10 to the fifth of the TP nll astrocytes into the recipient brains led to neurological morbidity. With a median survival of 22 days. Longitudinal imaging in vivo has been used to define tumor growth kinetics.

Therefore, in this experiment, TR penal astrocytes and engineered to express luciferase were injected into the brains of immune competent syngeneic. Litter mates and tumor growth was determined by serial bioluminescence imaging. A 15 fold increase in the bioluminescence was observed over a period of 16 days.

Following this procedure, drug testing can be performed in vitro and in vivo to determine efficacy and to test novel drug combinations. After watching this video, you should have a good understanding of how to harvest cortical astrocytes from non crea expressing conditional genetically engineered mice, how to induce genetic recombination in vitro, and how to model tumorgenesis using orthotopic allografts in immune competent mice.