Stereotaxic Transplantation of Neural Cell Suspension into a Mouse with Traumatic Brain Injury

Begin with a preloaded syringe attached to a stereotaxic syringe pump.

The syringe contains a human-induced pluripotent stem cell-derived neural cell suspension.

Position the needle above the surgically exposed brain cortex of a mouse with traumatic brain injury.

Lower the syringe to touch the dura with the needle tip, adjust the coordinates to target the injury site, and retract slightly. Now,

create a small incision, establishing an entry point for the needle.

Next, lower the plunger to allow the flow of the cell suspension.

Lower the needle and penetrate the brain to reach the gray matter-white matter border of the deep cortex.

Finally, slowly infuse the cell suspension while irrigating the surgical site with saline to maintain hydration.

Upon completion, withdraw the needle, re-irrigate, and close the incision.

Over time, the transplanted cells mature, restore neural circuits, and repair brain damage.

Roughly 24 hours after the craniectomy, place the mouse back into the stereotaxic frame.

Cover the mouse with the fenestrated surgical drape. Lavage the incision site with sterile saline to clean the site and to loosen the sutures. Use a 70% ethanol soaked cotton swab to gently sterilize the incision site and use fine tweezers and ophthalmic scissors to remove the sutures.

Then irrigate the surgery and craniectomy site with abundant sterile saline. In a cell culture biosafety hood, gently swirl or tap the tube of cells to ensure a homogenous cell suspension. Use a micropipette to load approximately 7.5 microliters of cells into a Hamilton syringe through the plunger end.

Holding the syringe at a 120 degree angle with the plunger end facing down, insert the plunger taking care not to introduce an air bubble between the suspension and plunger tip. Attach the gasket assembly to the pipette needle and attach the needle to the syringe. Push the plunger to move the cell suspension into the pipette needle and attach the syringe to the stereotaxic syringe pump.

Advance the plunger to make sure the syringe pump assembly is working properly and move the needle into the coordinates for injection. Align the needle tip to bregma and set the x and y-coordinates to zero. Move the needle tip over the craniectomy to two millimeters lateral and minus one millimeter posterior to bregma and touch the needle tip to the dura mater surface.

Raise the needle slightly then make a small incision in the dura mater at the location where the needle made contact. Set the stereotaxic coordinate to z equals zero and push the plunger to confirm that the cell suspension is flowing adequately before introducing the needle into the brain. Introduce the needle into the brain to a depth of z equals minus 1.4 millimeters to place the graft at the gray matter/white matter border of the deep cortex.

Start the syringe pump to infuse the cell suspension over a 15-minute period. Use a long working distance microscope to monitor cell suspension outflow irrigating the surgery site with sterile saline during the injection to maintain tissue hydration. When all of the cells have been delivered, slowly withdraw the transplantation needle and irrigate the surgery site with additional saline before closing the incision with new sutures.

Then deliver post-operative care as demonstrated.