The overall goal of this procedure is to create a platform for in vitro electrical stimulation of stem cells on a conductive polymer scaffold, with subsequent in vivo implantation. This method can help answer key questions in stroke research and stem cell biology by providing a means to modulate stem cells. The main advantage is that the stem cells can be optimized in vitro, and subsequently implanted.
Although this method can provide an insight into stroke recovery, it can also be applied to other systems as a new cell delivery method. Generally, users new to this technique will struggle due to the precision required for cell chamber assembly, and the technique involved with the craniectomy. In preparation, autoclave the 2.5 by 12.5 centimeter metal plates and the flow valves.
Next, using a chamber slide as a template, cut two pieces of PDMS. One piece serves to outline of the chamber's perimeter. On the second piece, cut out two adjacent pieces from the inside of the chamber slide.
Both pieces should be rectangular and match the chamber slide. Now, layer the materials. Start with the metal plate, then the uncut PDMS, then the polypyrrole plate, oriented perpendicularly, then the PDMS with cut-outs, and finally, the cell chamber.
Align the layers carefully, and clamp the apparatus together. Next, cut two lengths of a metal wire to extend from the apparatus to the outside of the incubator that will contain it. 60 centimeter lengths are usually long enough.
Next, use silver paste to attach a wire to each end of the polypyrrole plate protruding from the outside of the chamber. Once the silver paste is cured, reinforce and seal the connections using an epoxy. Now, record the resistance across the assembly using a multimeter.
The applied field must be the same in each chamber. Next, plate the cells onto the assembly, and electrically stimulate them as described in the text protocol. Prior to implanting the cells, remove the wires from the assembly.
Then, unclamp the cell chambers and separate the PDMS from the conductive scaffold. The scaffold is now ready for implanting. One day prior to the surgery, provide the subject animal ampicillin in its drinking water.
On the day of the surgery, anesthetize the rat with isoflurane, and confirm the anesthesia with a toe pinch. Now, apply artificial tears to the rat's eyes to keep them moist. After shaving the skull, drape the animal, and set up for a sterile surgery.
Wear protective garments, unpack the sterilized tools, and sterilize the scalp. For the craniectomy, drill a hole above the left cortex down to the dura mater. Make the opening slightly larger than 1 by 3 millimeters, the size of the implant.
Then, open the dura and remove the dura mater from the brain using a micro-thin surgical needle. Remove the rig from the TC-culture incubator and disable the rigs. Next, carefully implant the conductive scaffold from the in vitro system onto the rat cortex.
Then cover the implant with Surgicel to secure it, or it may move during the skin closure. Now, suture the wound shut using 4-0 silk suture. After completing the sutures, subcutaneously inject the rats with buprenorphine SR.Then, place the rat in a recovery cage until it regains consciousness.
The post-operative procedure is detailed in the text protocol. Human neural progenitor cells were exposed to electrical stimulation, and then stained with cell viability assays. The green color is indicative of a living cell.
The cells were clearly able to survive the electrical stimulation. Factors released from the neuro-progenitor cells that are important in stroke recovery were evaluated from one microgram of total RNA using quantitative RTPCR. After normalizing the results using the delta delta cycle threshold method, they reveal then the exposure to electrical stimulation up-regulated BDNF and THBS1 expression.
After watching this procedure, you should have a good understanding of how to create a cell culture system for electrically-conducting stem cells, and subsequent implantation for those stem cells into a rat model. Once it is mastered, the procedure can be done in 45 minutes if it is performed properly. While you're attempting this procedure, it is important to remember to always monitor the animals for proper breathing pattern and membrane color, to ensure proper anesthesia.
Don't forget that working with rats and stem cells can be hazardous, and precautions, such as wearing proper PPE, should always be taken.
