Surgical Transplantation of Tumor Cells into the Spinal Cord of Mice

The scope of our research is to develop a murine model of spinal cord glioma. We aim to answer questions like, how can the model's reproducibility be improved? What are the optimized surgical techniques for tumor cell implantation?

And how can this model assist in developing effective treatment for spinal cord glioma? The current experimental challenges mainly lie in three aspects. Firstly, practice defining the injection position in the spinal cord is difficult.

Secondly, minimizing cell split after the injection is crucial. Lastly, reducing physical damage to the spinal cord during the process requires refined operational skills. Our protocol addressed the research gap in existing murine spinal cord glioma models.

Current models often lack reproducibility and optimization in surgical techniques. We aim to fuel this by developing a more reliable model with precise injection methods and minimize physical damage to enhance research and treatment exploration. To begin, culture the GL261-luciferase cell line in the complete DMEM until the logarithmic growth phase.

Then wash the tumor cells twice with sterile PBS and incubate with 0.05%trypsin EDTA solution for three minutes. Transfer the resulting cell suspension into a new tube and centrifuge at 500G for five minutes. Discard the supernatant using a pipette and resuspend the pellet in PBS.

Next, stain the cells with trypan blue and count the viable cells using a cell counter. Prepare the cell suspension at a concentration of five times 10 to the power of six cells per milliliter in sterile PBS. To begin, place the anesthetized mouse on a surgical table.

Expose the skin and prepare a clean surgical window. After shaving the hair from the dorsal neck region, disinfect the skin with iodine solution, followed by wiping with 75%alcohol for deiodination. Position the mouse with its dorsal side facing upward and secure the limbs to the table using medical tape.

Place a one to two centimeter thick gauze pad under the neck area for support to provide better access to the spinal cord. Next, using a surgical scalpel and blade, make a longitudinal incision approximately 1.5 centimeters along the neck. Gently separate the neck muscles using blunt dissection, while avoiding injury to any blood vessels.

Now carefully dissect the muscles adjacent to the cervical vertebrae to expose the seventh cervical vertebral spinous process, a distinct bony landmark in mice. Set the puncture point at 0.5 to 0.9 millimeters from the midline of the spine. Rinse a 10-microliter flat needle syringe thoroughly with sterile PBS two to three times.

Draw two microliters of the tumor cell suspension into the syringe without introducing air bubbles. Stabilize the cervical vertebral spinous process by gently grasping and lifting it with forceps. Use a beveled needle to puncture the dura mater.

Then switch to a flat-needle syringe to inject the tumor cells. After injection, close the skin incision by suturing with a 3/0 nylon suture. 10 days post-surgery, mice inoculated with GL261-luciferase cells exhibited hind limb weakness and significant weight loss of approximately 20%with decreased survival rate, progressing to hind limb paralysis with tumors, confirmed through bioluminescent imaging, gross visualization and histological examination.

The B16-F10 melanoma model displayed similar tumor progression to GL261 with bioluminescent imaging and gross visualization, confirming spinal cord tumor establishment along with weight loss and mortality within 18 days. Postoperative analysis showed mild spinal cord injuries resolving within three hours in 54 mice and moderate injuries in three mice resolving within three days. Mice with moderate injuries showed more significant body weight loss compared to those with mild injuries.