膠芽腫の治療の幹細胞シード足場の頭蓋内注入画像誘導切除

The overall goal of this procedure is to surgically resect brain cancer in mice and to implant a scaffold seeded with tumor-homing therapeutic stem cells into the postoperative resection cavity. This method can help answer key questions in the cell therapy and neuro-oncology fields, such as how surgical resection affects cell implantation into the surgical cavity, how effective new therapeutic agents are on postsurgical brain cancer, and how scaffold-based delivery systems influence therapeutic outcomes. The main advantage of this technique is that brain cancer interventions can be studied in mice in a way that closely mimics the clinical standard of care in human patients.

Though this method can provide insight into stem cell therapy delivered on scaffold, it can also be applied to other interventions, such as nanoparticles, small molecules, oncolytic viruses, or other emerging therapies. Prepare the scaffolds 48 hours prior to implantation in mice by first cutting PLA scaffolds into resection cavity-sized pieces of approximately two by two millimeters. Sterilize the scaffolds by immersing in 70%ethanol for 15 minutes.

After 15 minutes, immerse the scaffolds in PBS. Then place scaffolds in Dulbecco's modified Eagle's medium containing 10%fetal bovine serum and 1%penicillin-streptomycin while preparing cells for seeding. To prepare the cells, first lift cultured MSCs using 0.05%trypsin.

Incubate at 37 degrees Celsius for five minutes. Following the incubation, ensure that the cells have lifted. Then add DMEM to the flask to inactivate the trypsin.

Next, transfer flask contents to a 15-milliliter centrifuge tube. Count the cells using a hemocytometer. Then pellet five times 10 to the fifth MSCs for each scaffold via centrifugation at 100 times g for five minutes.

Following the centrifugation, aspirate the supernatant, and resuspend the MSCs in five microliters of DMEM per five times 10 to the fifth cells. Then remove a scaffold from DMEM immersion with forceps, and place it on the lid of the six-well plate. Lift the scaffold off the lid, leaving behind a droplet of excess DMEM.

Place the scaffold back on the lid again in a new, dry location. Repeat three to five times. Then place the partially dried scaffold in a new six-well plate for seeding.

A partially dried scaffold provides optimal cell seeding results. If the scaffold is too wet, cells will slide off the scaffold and adhere to the plate below. If the scaffold's too dry, the droplet will not spread over the entire scaffold, resulting in poor initial cell distribution.

Using a pipette, gently mix the tube of MSCs to homogenize the suspension, as some cells may have settled to the bottom. Slowly pipette 2.5 microliters of freshly mixed MSC suspension directly on the scaffold, creating a small droplet over the top of the scaffold. Add 300 microliters of DMEM to edges of each well to prevent rapid evaporation of the cell droplet.

Incubate at 37 degrees Celsius for 30 minutes, allowing the cells to attach to the scaffold. Following the incubation, use forceps to gently flip the scaffolds over in the well plate. Seed 2.5 microliters of freshly mixed MSC suspension to give a total of five times 10 to the fifth cells seeded per scaffold.

After an incubation of 30 minutes to allow the cells to attach to the scaffold, cover the scaffolds in DMEM by adding two milliliters to each well of the six-well plate. Gently lift the scaffolds to allow media to flow underneath them. Incubate at 37 degrees Celsius in this state for 48 hours prior to implantation surgery.

Begin by placing the stereotaxic frame on the stage of a fluorescence dissecting stereomicroscope. Then secure the anesthetized mouse in 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 proper control it, 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 one microliter of fibrinogen followed by one 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.

These phase-contrast, fluorescent, and combined images show U87 cancer cells engineered with lentiviral constructs to express mCherry and firefly luciferase markers. These phase-contrast, fluorescent, and combined images show corresponding images of stem cells engineered to express diagnostic GFP-Renilla luciferase or therapeutic GFP-TRAIL seeded onto the PLA scaffolding material. This brightfield and fluorescent overlay image shows the location of the tumor.

Here, the postsurgical resection cavity with remnant tumor foci is seen. This image shows the implanted PLA scaffold seeded with MSC-TRAIL. Here, postmortem brain tissue is seen with the scaffold overlaid.

This fluorescent overlay highlights the GFP-TRAIL cells. Once mastered, this technique can be done in under 30 minutes for each mouse, if it is performed properly. While attempting this procedure, it's important to remember that there will be intrinsic variability in the extent of resection achieved from mouse to mouse, similar to what is seen in clinics.

After watching this video, you should have a good understanding of how to perform a surgical resection of a brain tumor and evaluate its impact on therapeutic interventions that include scaffold-based stem cell therapy.