The overall goal of this procedure is to record the effect of the applied electric fields on neural progenitor cell migration in 2D culture and 3D ex vivo models. This is accomplished by first isolating and expanding neuron neural progenitor cells in vitro. The second step is to prepare a single cell suspension of neural progenitor cells.
Next, for 2D culture preparation, neural progenitor cells are plated in custom designed electro tactic chambers. Alternatively, for 3D ex vivo model preparation, neural progenitor cells are labeled with hooks 3, 3, 3, 4, 2, and injected into a spinal cord slice organotypic model. Ultimately, an electric field is applied to the electro chambers and time-lapse recording is performed to show that progenitor cells migrate towards the cathode of the electric field in both 2D and 3D environments.
The main advantage of this technique is that it provides a novel method for the live tracking of electric field induced migration of individual cells following transplantation into a 3D organotypic model, closely resembling the natural environment. This method can not only provide inset into the electro technique, migration of neuro ental cells transplanted into spinal cord slice, but can also be applied to the other systems that as brain slice models small. To begin this procedure, prepare 22 by 11 millimeter strips of glass by dividing the autoclave 22 by 22 millimeter.
Number one thickness cover slips in half using a diamond pen. Next, create a freestanding glass well of interior dimensions, 22 by 10 millimeters by gluing four vertically, standing 22 by 11 millimeter strips together with high vacuum silicone grease, allow the well to completely dry and harden overnight. The next day, attach 2 22 by 11 millimeter glass strips parallel to each other, leaving a gap of 10 millimeters to the base of a 100 millimeter culture dish using silicon grease.
Then seal off the region between these strips by attaching a 22 by 22 millimeter cover slip with silicone grease. Place the glass well onto the cover slips so that the interior walls create a confined space for seeding the cells. Waterproof all the joints with silicone grease.
Coat this confined region sequentially with poly de lycine, followed by laminin as described in the accompanying manuscript. In this procedure, obtain the brains from E 14 to E 16 mice and place them in cold D-M-E-M-F 12 basal medium. Next, remove the meninges under an anatomical microscope.
After that, transfer the brains to a 35 millimeter Petri dish. Use the fine forceps to dissociate the brains into tissue fragments. Then transfer them to a 15 milliliter tube centrifuge.
The samples at 800 RPM for three minutes to remove D debris. Next, A-D-M-E-M-F 12 containing BFDF and DGF to the samples. Tritrate ate them with a one milliliter pipette.
Then obtain the single cell suspension with a cell strainer. Plate the cells into the flasks at 20, 000 to 50, 000 cells per milliliter. Then perform a full medium change every three days, which involves centrifuging the cells at 1, 300 RPM for five minutes.
Discarding the supinate Resus, suspending the PT in fresh medium, and returning the cells to the flask the cells every six days after at least fives. Transfer the NEUROSPHERES to a 15 milliliter tube centrifuge at 1, 500 RPM for five minutes. Discard the supinate.
Re suspend the pellet in trips in EDTA to produce a single cell suspension. Before performing a cell count, prepare one milliliter of suspension containing 10, 000 cells. Next, remove laminin from the glass well and allow it to dry completely.
Replace it with one milliliter of cell suspension. After that, place the dish in the incubator at 37 degrees Celsius for a minimum of four hours to allow for attachments. Once the cells are sufficiently cofluent, remove the medium from the chamber.
Carefully remove the glass. Well then form a roof over the cells by carefully attaching an autoclave 22 by 22 millimeter glass cover slip between 2 22 by 11 millimeter strips with silicone grease. Remember to cover the cells with a few drops of medium to avoid drying out.
Then form an isolated medium reservoir at each end of the chamber by creating two watertight silicone grease barriers that run from one end of the dish to the other. Fill the chamber with fresh medium, ensuring a flow through from one medium reservoir to the other. Afterward, return the dish to the incubator for 12 hours to allow for cell recovery.
In the next step, prepare a lid to cover the dish by drilling two holes, one positioned over each reservoir of the migration chamber. Then replace the medium in the chamber with cultured medium containing 25 millimeter hippies buffer. After that, transfer the dish to the temperature controlled imaging system.
Set the experimental parameters for time lapse and multi-position recording. Align the chamber so that the cathode and anode are on the left and right respectively to ensure that the electric field vector runs horizontally as viewed under the microscope and recorded in the imaging system. Next, fill two beakers.
With Steinberg's solution. Connect a separate beaker to each medium reservoir using the pre-prepared glass bridges filled with 2%Steinberg's agar solution. Then place the silver electrodes connected to a DC power supply into the Steinberg solution.
In each beaker, set the voltage dial on the power supply to zero and switch it on. Measure the voltage across the electro tactic chamber with a voltage meter while turning up the voltage dial. Then adjust the voltage to suit the experimental requirements.
After that, start the time-lapse recording. Perform a medium change and readjust the voltage as required. Every hour, fresh, medium drugs or chemical agents may be added to the reservoirs as required.
Using additional holes drilled into the culture dish lid, one situated over each medium reservoir. In this step, dissect the lumbar spinal cords of two week old mice. Then slice the spinal cords into 500 micron thick sections with a machel wane tissue chopper under an anatomical microscope.
Separate the slices and select the ones with intact sagittal axial spinal cord structure. Next plate the slices in a 100 millimeter Petri dish containing 30 microliters MA gel. Place them as close to the center as possible.
Maintain them in a 5%carbon dioxide incubator at 37 degrees Celsius for at least 30 minutes. For the matri gel proteins to self-assemble, to produce a thin film that covers the surface of the spinal cord slices, it is very important that the matri gel has been assembled completely. Incubate the Petri dish at 37 degrees Celsius for another 30 minutes if necessary, then add four to six milliliters of D-M-E-M-F 12 medium containing 25 millimolar heis buffer, and 15 to 20%fetal calf serum.
Very gently to avoid medium flow directly onto the slices, ensure that the slices are not entirely submerged in medium, leaving the surface of the eggplants well exposed to the air. Change the medium twice weekly. Now incubate the neural progenitor cell suspension in the medium with five micromolar hooks.
3, 3, 3, 4, 2 for 30 minutes. Under a microscope, use a capillary glass tube to micro inject two microliters of suspension into the spinal cord slice. Slowly make sure that the capillary glass tube passes through the matrigel and reaches the insides of the spinal cord slice.
Keep the glass tube inside the spinal cord slice for at least 30 seconds to avoid cell suspension. Run over, then place the Petri dish containing the spinal cord slice into the incubator at 37 degrees Celsius with 5 cent carbon dioxide overnight. Next day, construct an electro tactic chamber around the spinal cord slice containing hooks 3 3 3 4 2 labeled neurogenesis cells before applying an electric field measuring 500 millivolts per millimeter.
Here the neural progenitor cells show highly directed migration towards the cathode when exposed to the electric fields, red lines and blue arrows represent the trajectories and direction of cell movement respectively. And here is the plot of the migration paths shown. Here are the neural progenitor cells labeled with hooks 3 3, 3, 4 2.
They were transplanted into the organotypic spinal cord slice at the starting point of the electric field treatment. After the neural progenitor cells have migrated directionally towards the cathode for 2.5 hours, the polarity of the electric field was reversed. The altering electric field polarity triggered a sharp reversal of axxis towards the new cathode.
Here is an image of the transplanted neural progenitor cells within the spinal cord slice. At the end of the time-lapse recording and a 3D reconstruction of the transplanted neural progenitor cells within the spinal cord slice is shown here. Mass mastered.
This technique can be done in two weeks if it's performed properly After its development. This technique now paves the way for researchers in the field of cell biology to explore the migration of neural progenic cells in 2D and 3D culture systems in response and apply to electric field.
