Fluorescence-guided Tumor Resection and MSC-seeded Scaffold Implantation in Murine Model: A Surgical Procedure to Deliver Therapeutic Mesenchymal Stem Cells into Brain Tumor Resection Cavity in Mouse Model

Begin by placing the stereotaxic frame on the stage of a fluorescence dissecting stereomicroscope. Then, secure the anesthetized mouse on the stereotaxic frame with continuous inhaled anesthesia supply via an isoflurane nose cone adapter. Maintain body temperature with a heating pad. Apply ophthalmic ointment to the eyes to prevent drying of the cornea. Then, sterilize the incision site of the scalp with a series of three alcohol and three betadine wipes.

Perform the toe pinch reflex test on each limb and confirm negative response to ensure proper anesthetization. Using forceps, pinch and gently lift the scalp. After making a midline linear rostral-caudal incision with surgical scissors, irrigate the incision site with PBS and remove subdermal fat with a cotton tip applicator in a circular brushing motion.

Arrange the skin so that the previously established cranial window is fully visible. Use an 18 gauge needle to gently puncture the dura just interior to the borders of the cranial window. Repeat until the incision fully traces the interior of the window. Remove the dura by peeling it away using fine forceps, revealing the underlying parenchyma and tumor.

Next, dim the room lights and turn the stereomicroscope fluorescence mode on. Locate the U87 mCherry and firefly luciferase-expressing tumor. Load a 200-microliter pipette tip into the end of the tubing of a vacuum pump. Then, turn on the vacuum pump. Resect the tumor by gently aspirating the fluorescent tissue until no signal remains. To control bleeding, irrigate with cold PBS and apply steady pressure with a cotton tip applicator.

Use a combination of saline, steady pressure with a cotton tip applicator, and/or a hemostatic agent, such as SURGICEL, to stop bleeding prior to implanting the scaffold. If not properly controlled, a potentially dangerous hematoma may form. After resection, turn off the vacuum pump and discard the pipette tip. Turn the fluorescence off and the room lights back up. Immediately prior to implantation, slowly dip an MSC-seeded PLA scaffold in PBS to remove unwanted media and associated components. Implant the scaffold into the resection cavity.

If needed, add 1 microliter of fibrinogen, followed by 1 microliter of thrombin to secure the scaffold in place. With the dura removed and the bone flap from the cranial window already discarded, seal the wound by closing the skin and applying surgical glue. Remove the animal from inhaled anesthetic and allow it to recover on a heated surface.