Cell-based therapy has huge potential for brain regeneration. The protocol that we demonstrate here is valuable, as it allows longitudinal in vivo imaging of transplanted cells in the mouse brain. This protocol is especially useful to gain insights about the migration and the survival of the graft in the same animal over a period of time.
This procedure can be applied to any luciferase-expressing cell line transplanted to the mouse brain. However, the signal strength may vary depending on the depth of the transplantation or the luciferase expression in the respective cell line. Demonstrating the procedure will be Rebecca Weber, an excellent PhD student in our laboratory.
Begin by collecting a vial of cells from minus 150 degrees Celsius storage and transfer it to the laboratory. Quickly transfer the vial to a water bath tempered at 37 degrees Celsius for two to three minutes until ice crystals dissolve. Then, transfer the vial to the biosafety cabinet and pipette the whole content into a sterile 15-milliliter conical tube.
Add nine milliliters of sterile PBS and centrifuge at 300 G for five minutes at room temperature. Remove the supernatant by aspiration without disturbing the pellet and count the cells before the final spin using an automated cell counter. Transport the animal from the induction chamber to the stereotaxic frame, maintaining anesthesia using a face mask.
Apply ophthalmic lubricant to prevent the eyes from drying out. Shave the mouse scalp with an electric razor and disinfect the skin with 5%betadine solution using cotton swabs. Secure the mouse head and insert the ear bars into the external meatus.
Drill a hole with a diameter of two to three millimeters through the skull with a surgical dental drill. Resuspend the prepared cells in the tube and draw two microliters of cell suspension into a syringe. Place the syringe above the target site and slowly move the needle to the surface of the dura and calculate the depth coordinates.
Double click the Living Image software icon and select a user ID from the dropdown list. Then, click Initialize in the Control Panel that appears. In the Living Image software, check the Luminescent and Photograph boxes in the Control Panel and select Auto exposure.
Select a field of view. Enter the subject height and select the use subject height focus option. Manually set the filtering parameters including large Binning, F/Stop, blocked Excitation Filter, and open Emission Filter.
Inject the luciferin intraperitoneally, and after five minutes, anesthetize animals with a continuous isoflurane supply. Shave the sedated animals on the head region using a conventional hair shaver. Place the animal in the imaging chamber and start imaging 15 minutes after the luciferin injection by clicking Acquire in the Control Panel.
Before transplanting neural progenitor cells in the mouse brain, the cells were tested for successful transduction by expression of EGFP in vitro. The successful transplantation was confirmed by the presence of the red Firefly luciferase signal. After transplantation of 6, 000 to 180, 000 cells in the right sensory motor cortex of the mouse, bioluminescence signal was detectable at all concentrations.
The cells survived for at least five weeks after transplantation. The transplanted cells were successfully detected ex vivo in a subsequent histological analysis through the EGFP reporter and immunostaining with anti-human nuclei. It's very important to carefully calculate the injection coordinates to avoid missing the target site.
And also, leave the syringe in place for at least five minutes after transplantation to avoid any reflux. After in vivo bioluminescent imaging, the brain tissue can be either prepared for histological analysis, or the transplanted cells can be isolated using FACS and characterized with different or mixed technologies. Overall, this technique provides a quantitative and continuous tracking of transplanted cells in the brain, and it can be applied to a huge number of neurological diseases including stroke and traumatic brain injury.
