This method is useful to order to analyze the role of some molecules in axonal guidance and neuronal migration during neuro system development. The main advantage of this technique is that the analysis of the results is quick and simple and doesn't require complex software which would imply a lot of training for researchers. Demonstrating the procedure will be Mirian Segura-Feliu, a technician from our laboratory and myself.
To begin plate 200, 000 COS-1 cells into 35 millimeter Petri dishes and incubate with complete culture medium in a cell culture incubator in order to read 70 to 80%confluency overnight. On the following day, to transfect COS-1 cells with the DNA encoding the candidate molecule using liposome based transfection method. First mix 250 microliters of serum free medium and one to 2 micrograms DNA in a 1.5 milliliter centrifuge tube and incubate at room temperature for five minutes.
To prepare a liposomal tube, add 240 microliters of the serum free medium and 10 microliters of liposomal transfection reagent, and incubate at room temperature for five minutes. After incubation, add the content of the DNA tube to the lipsomal tube. Gently mix and incubate at room temperature for 15 minutes.
Replace the medium on the cultured cells with 1.5 milliliters of the serum free medium. Add the DNA liposomes mixture slowly and drop wise to the cells. Incubate in the carbon dioxide cell culture incubator for three hours.
After completed incubation, replace the medium with the complete culture medium and incubate overnight in the incubator. On the following day, rinse the cells with 0.1 molar Dulbecco's PBS. Add 800 microliters trypsin EDTA per each dish and incubate for five to 15 minutes in the carbon dioxide incubator.
Collect detached cells with two milliliters of complete culture medium into a 15 milliliter centrifuge tube and add 10 milliliters of the same medium. Centrifuge the cells at four degrees celsius at 130 times g for five minutes. Remove the supernatant and preserve the pellet containing COS-1 cells on ice.
After preparing collagen working mixture, add 100 to 150 microliters of the collagen mixture to the pellet of the transfected cells and mix gently by pipetting up and down. Spread 45 to 50 microliters of collagen cell pellet mixture onto a 35 millimeter Petri dish to form a uniform band of collagen cells, approximately one to 1.5 centimeters long. Place the dish in the incubator at 37 degrees celsius and 5%carbon dioxide until the gelation is observed.
Prepare a second strip containing control cells in a second culture dish and place it in the incubator. When gelation is completed, add three to four milliliters of 37 degrees celsius warmed COS-1 complete culture medium to each dish containing the gelled collagen cell strips and place them in the incubator. Use a fine scalpel or a tissue chopper to cut the collagen cell strips to generate small square pieces, 400 to 500 micrometers in length.
Transfer all the sections from the same transfection condition to a Petri dish containing three to 3.5 milliliters of neuronal culture media. Check under a dissecting microscope the quality of the pieces and place the dishes in the incubator. Use scissors to cut embryo horns on embryonic day 16.5 from the abdominal cavity of a sacrificed pregnant female rat and place them into a large Petri dish containing cold HBSSg buffer.
Place the dish in the laminar flow hood and use straight forceps to extract the embryos and place them into a new dish containing cold HBBSg. With small forceps, remove the skin, and then with curved and straight forceps, carefully dissect the brain and place into a dish containing cold HBSSg. Under a dissecting microscope, using a scalpel or fine scissors, cut the brain in half along the midline to separate both the hemispheres.
Then, remove the diencephalon and with fine forceps, remove meninges and blood vessels from the brain pieces. Use 100%ethanol to clean all parts of the tissue chopper, especially PTFE cutting plate and the razor blade and keep the tissue chopper in the laminar flux hood under UV illumination for 15 minutes. Transfer each tissue piece to the cutting plate of the tissue chopper.
After the tissue has been chopped, transfer the pieces from the 35 millimeter Petri dishes filled with three to four milliliters of complete NCM. Using fine tungsten needles, finish the tissue dissection in complete NCM. Check the quality of the obtained slices under the dissecting microscope, making sure that the layers are clearly identifiable in the dark field optics and dissect the region of interest with tungsten needles.
Keep the good quality pieces in complete NCM medium in the carbon dioxide incubator. Visual demonstration is critical in the steps related to the preparation of the co-cultures which facilitates the words to understand the process. Place several sterile four well culture plates in the laminar flow hood and prepare a collagen working mixture as previously.
Add 15 to 20 microliters of the hydrogel mixture into the bottom of each well to produce a circular collagen base. Place the dishes in the incubator until complete gelation is observed. And check the quality of the gelled collagen.
With a pipette, transfer a small piece of COS-1 cell aggregate onto the hydrogel base, then use a pipette to place a tissue piece on the same base, close to the piece of cells aggregate at a distance of one explant size. After preparing a new working collagen mixture on ice, gently pipette 15 to 20 microliters of this new mixture to cover the explant and the cell aggregate, making a sandwich like hydrogel culture. Use a fine tungsten needle to reorientate the explant so it faces the cell aggregate at 500 to 600 micrometers.
Return the plate to the incubator until the gelation is observed and then add 0.5 milliliters of complete NCM supplemented with 2%B27 supplement and keep cultures for 36 to 48 hours in the incubator. When hippocampal axons were confronted with Netrin-1, they grew preferentially towards the source of Netrin-1, indicating that Netrin-1 acts as a chemo attractive molecule for these axons. In the control condition, or mock transfection, all axons grew radially, without any directional preference.
When hippocampal axons were confronted with Sema3E secreting cells, most of them grew opposite of the cell aggregate, indicating that Sema3E acts as a chemo repulsive molecule for these axons. This schematic representation of the axonal response and quantification method shows that in control conditions, the axons were equally distributed in both proximal and distal quadrants, shown with radial outgrowth. This indicated a proximal distal or PD ratio of one.
When explants showed increased number of axons in the proximal quadrant in comparison to the distal, indicating chemo attraction, the PD ratio was greater than one. When the number of axons was higher in the distal quadrant than in the proximal one, indicating chemo repulsion, the PD ratio was lesser than one. Following this method, researchers can obtain essential information about the role of some molecules during neuronal development, which allows us to establish a starting point to further explore the functions.
Although this technique is useful in neuronal development as this is, it could also be applied in pharmacological screening, angiogenesis, and tissue engineering as strategies.
