マウスモデルにおける膠芽細胞腫に対する治療用幹細胞の鼻腔内送達

The overall goal of this protocol is to deliver therapeutic stem cells to the brain intranasally in a preclinical mouse model of glioblastoma. This method can help answer key questions in neuro-oncology field about whether therapeutic stem cells treatment can achieve a robust, therapeutic effect in pre-clinical brain tumor model. The main advantage of this technique is that intranasal administration facilitates the delivery of non-invasive, repeatable brain tumor treatment that bipasses the obstructive blood-brain barrier.

This technique is not limited to glioblastoma. It can be broadly extended toward therapy of other brain malignancies and brain disorders. My colleagues, Dou Yu, a research assistant professor, Gina Li, a research technologist will be demonstrating this procedure.

Begin by culturing the glioma cells of interest from frozen stock at one times 10 to the six cells per T 75 square centimeter culture flask in 15 milliliters of serum supplemented or serum-free medium as appropriate in a humidified, carbon-dioxide-supplemented cell culture incubator. When the cultures have reached 80 percent confluency, wash the culture with calcium and magnesium-free PBS. Then, dissociate the cells with point zero five percent trypsin at room temperature.

Then tap the flask to detach the cells from the bottom on the container. Immediately neutralize the enzymes with eight to nine milliliters of serum-containing medium. Use serological pipettes to transfer the resulting cell suspension into conical tubes.

Collect the cells by centrifugation. Followed by two washes in 10 milliliters of PBS each. We suspend the pellets in sterile saline at a five to 20 times 10 to the fifth cells per two point five microliters concentration and transfer the cells into a sterile micro-centrifuge tube on ice.

After confirming the appropriate level of sedation by toe pinch, mount an approximately six-week old, immuno-compromised mouse onto an ethanol-sterilized stereotaxic frame in the appropriate head position. Put the inserted Hamilton micro-syringe as perpendicular to the surface of the skull around the burr hole. Use the stereotaxic frame knobs to position the flat tip of the micro-syringe until it is just at the opening of the burr hole then slowly lower the syringe until the needle reaches three millimeters below the dura surface.

Retract the syringe zero point five millimeters to maintain the needle tip at two point five millimeters below the dura and inject two point five microliters of pre-loaded glioma cells over a one minute period. Leave the needle in position for another one to two minutes before slowly withdrawing the syringe over the course of one minute to minimize the cell backflow. Apply a sterile bone wax to avoid extra-cranial tumor add growth and use seven millimeter stainless steel wound clips to close the wound.

Then deliver one milliliter of 37 degree Celsius sterile lactated ringer solution by subcutaneous injection and place the animal in a recovery chamber positioned halfway onto a heating pad with monitoring until full recovery. Seven to eight days post glioma cell implantation, preform head-only exposure to the radiation path. Then deliver two grades of irradiation to the animals everyday for five consecutive days, assessing the tumors by bioluminescence imaging after the last irradiation dose.

For human neural stem cell genetic modification, when the stem cell culture reaches 75 to 80 percent confluency, adhering CXCR4 encoded antivirus at five viral particles per cell in polybrene directly into the medium for eight to 16 hours of culture. At the end of the incubation, replace the transduction medium with fresh blasticidin supplemented medium for positive selection of the transduced cells. Harvest the cells by trypsin digestion as demonstrated.

Validate the level of CXCR4 expression by flow cytometry using anti-CXCR4 anti-body and matching isotope anti-body controls according to standard flow cytometric analysis protocols. Then suspend the cells in sterile saline for intranasal delivery. For hypoxic preconditioning of the neuro cells, when the culture reaches 90 percent confluency, transfer the flask to a humidified carbon dioxide incubator supplied with one percent oxygen.

After 24 hours, harvest the cells by trypsin digestion. Suspend the cells in sterile saline for intranasal delivery. To load human neural stem cells with oncolytic virus, infect the stem cells with 50 conditional replicative adenovirus viral particles per cell, according to standard adenovirus transfection protocols, for two hours at room temperature with periodic tapping.

Then wash the cells three times with fresh medium to remove any of the unbound viral particles and suspend the cells in sterile saline for intransal delivery. When the cells have been modified as experimentally appropriate, place the anesthetized, tumor-bearing animals in the supine position on a clean drape over a heat pad and place a padded pillow under the heads to maintain the nostrils at an upright angle. The pillow material should be either alcohol-wipe-able plastic or disposable rolled up paper towels to avoid cross-animal contamination.

When the mice are in position, use a micro-pipette to dispense two microliters of the experimental human neuro stem cell population of interest into each nostril of the first mouse. Visually confirm an aspiration of the droplet before applying the next volume of cells. Repeat the delivery to each mouse in turn.

Then wait five minutes and apply the next round of cells. When all four rounds of cells have been administered, place the animals in a recovery chamber with monitoring until full recovery. Both hypoxic pre-treatment and CXCR4 overexpression significantly upregulate the cell membrane presence of CXCR4 receptors as demonstrated by flow cytometry.

The presence of micron-sized paramagnetic ion oxide labeled stem cells in the tumor can be confirmed via Prussian Blue Staining. The tumor tropism demonstrated by neural stem cells can be visualized by histological analysis of the tumor tissue. Tissue analysis also reveals that head-only irradiation induces a reduction in tumor volume as well as SDF-1 upregulation in tumor and surrounding tissues.

Confirming that CXCR4 receptor upregulation in neural stem cells, we facilitate tumor tropism after irradiation. Survival analyses indicate that although irradiation alone is beneficial for the survival of tumor-bearing animals, additional benefits can be achieved using hypoxic neural stem cells loaded with irradiated oncolytic virus over control-irradiated, non-hypoxic stem cells loaded with oncolytic virus. Significant survival benefits are also observed with CXCR4 transduced neural stem cells after oncolytic viral loading and irradiation.

Further, both hypoxic neural stem cells and CXCR4 transduced neural stem cells exhibit a significantly enhanced tumor-targeted delivery of oncolytic virus particles as evidenced by viral hexon protein staining. Once mastered, this intranasal delivery technique can be completed in one hour if it's performed properly. While attempting this procedure, it's important to remember to follow all the steps in a timely manner exactly as described.

This technique allows the exploration of therapeutic potential stem cells for broad spectrum brain disorders in pre-clinical rodent models. After watching this video, you should have a good understanding of how to perform consistent and effective intranasal therapeutic stem cell delivery for the targeting of mouse brain tumors. Don't forget that working with human cells and viruses can be hazardous and that precautions, such as using a-subject techniques and proper personal protection equipment, should always be taken when performing these procedures.