The overall goal of this procedure is to seed, expand and differentiate neural stem cells that were prepared from the hippocampus of adult non hibernating yearling arctic ground squirrels. This is accomplished by first seeding the A GS neural stem cells. Next, expand and seed the neural stem cells into differentiation medium.
The third step of the procedure is to differentiate and maintain the neurons, and finally, the cells are fixed or treated and fixed for analysis. Ultimately, results can be obtained that show a GS neuronal progenitors continue to divide through two weeks of differentiation, maintenance, and even under ischemic conditions. Neuron numbers are determined by staining and fluorescent microscopy techniques that measure neuron ratios.
The implications of this technique extend toward the discovery of therapies for stroke and cardiac arrest. Since arctic ground squirrel, differentiated neurons are tolerant to all ischemic insults Prior to starting this procedure, prepare the flask to be used in A-G-S-N-S-C expansion by coating them with polyol ornithine. Follow the Polyol Ornithine working solution by leaving it in the incubator.
Once thawed, the solution could be stored for up to one month at two to eight degrees Celsius. Add eight milliliters of Polyol ornithine working solution to a vented T 75 flask and evenly distribute the solution over the entire culture surface of the flask. Incubate the flask at 37 degrees Celsius for a minimum of 24 hours, preferably in an incubator without added CO2 prior to use.
Aspirate the Polyol ornithine working solution from the flask and wash twice with sterile tissue culture. Grade water aspirate the liquid until the flask is nearly dry before you use to thaw and seed arctic ground squirrel neural stem cells. First, remove a vial of A-G-S-N-S-C from the liquid nitrogen doer and check that the vial cap is securely sealed.
Hold the top of the vial while leaving its bottom half in a 37 degree Celsius water bath for approximately 60 to 90 seconds. Keep the vial cap out of the water to avoid potential contamination. Remove the vial from the water bath when it is almost completely thawed.
A small amount of ice should still be visible. Do not over thaw as doing so may damage the cells. Spray the exterior of the vial with 70%ethanol or isopropanol, and then take the vial to a biological safety cabinet.
Remove the cap carefully to avoid contamination or splatter. Using a sterile pipette. Add one milliliter of basal medium into the vial and gently resus.
Suspend the cells. Transfer the resuspended cells to 25 milliliters of warm basal medium in a 50 milliliter centrifuge tube. Centrifuge the cells at 150 G for six minutes while the cells are spinning down.
Add 10 milliliters of expansion medium to the dry poly L ornithine coated T 75 flask, and then add 40 microliters of Rh FGF basic When centrifugation is complete. Aspirate the sate and fluid carefully without aspirating the pellet.Resus. Suspend the cells in 10 milliliters of expansion medium.
Transfer the resuspended cells into the media in the T 75 flask. Gently rock the flask side to side to evenly distribute the cells within the vessel. Place the seeded flask in a 37 degrees Celsius 5%carbon dioxide incubator.
Cells should attach to the flask and begin to form processes within two hours of seeding on the day following inoculation. Spike the flask of A-G-S-N-S-C with 40 microliters of Rh FGF basic. Two days after thawing and seeding cells perform a full medium replacement using 20 milliliters of expansion medium plus a 40 microliter spike of RH FGF basic on the third or fourth day after inoculation.
The A-G-S-N-S-C should be passaged aspirate the medium for the T 75 flask and add 2.5 milliliters of trypsin EDTA swirl gently to ensure all cells are coated with the trypsin. EDTA and gently tap the culture vessel from several sides to encourage detachment. Once the cells have become detached, add 10 milliliters of basal medium to the flask, gently swirl to ensure all the trypsin solution is neutralized.
Pipette the cells into a sterile centrifuge tube containing 10 milliliters of basal medium. Then wash the flask with an additional 10 milliliters of basal medium and combine it with the first wash centrifuge the cells at 150 G for six minutes. After centrifugation, the cells should form a clean, loose pellet, aspirate the S natin from the centrifuge tube.
Then using a 10 milliliter pipette, add 10 milliliters of prewarm basal medium, and resus. Suspend the cell pellet by gently pipetting up and down two times. If this method is followed closely, a yield of three or more doublings may be expected giving a population of greater than 4 million cells from an initial number of approximately 500, 000, we will demonstrate the inoculation of A-G-S-N-S-C into a poly L lysine coated 96 Well plate for differentiation, although any multi-well format can be used for this procedure.
First, determine the volume of cell suspension needed for your experiment and the amount of FBS to be added to the A-G-S-N-S-C differentiation medium to achieve a final concentration of 2%FBS. An example of these calculations is shown in the protocol text next in a biosafety cabinet. Mix the FBS and differentiation medium.
Then carefully mix the cells with a calculated volume of differentiation medium with FBS and transfer cells in the medium to a sterile combi tip reservoir. Using a multichannel pipette and large orifice tips, dispense 200 microliters of diluted cell suspension from the reservoir to each well at the poly L lysine coated 96 well plate incubate the plate for one and a half to two hours at 37 degrees Celsius, 5%carbon dioxide to allow the cells to attach. The medium.
Change can be a challenging part of this technique because you may actually injure the cells using regular sized pipette tips. Therefore, we use large orifice pipette tips so as not to injure the cells during the medium. Change When the cells have attached carefully remove 50%which is 100 microliters in this case of the medium from each well using large orifice pipette tips.
Carefully replace the volume removed with warm differentiation medium. Again, using large orifice pipette tips. Incubate the plate at 37 degrees Celsius, 5%carbon dioxide two days after inoculation.
Perform a 50%medium change using differentiation, medium and large orifice pipette tips. The neuronal maintenance medium for maintenance of neurons should be prepared weekly following the manufacturer's instructions. Two days after the last differentiation.
Medium change. Perform a 50%medium replacement using neuronal maintenance. Medium carefully remove 100 microliters of the differentiation medium from each well using large orifice pipette tips and replace with 100 microliters of neuronal maintenance medium.
Continue to perform 50%medium replacements using neuronal maintenance medium every two to three days. Neuronal processes should appear within a few days of changing to neuronal maintenance medium. The cell should be used within three weeks of being transferred to neuronal maintenance.
Medium arctic ground squirrel. Adult neural stem cells will continue to double every 24 hours for three to five days for at least two passages when seated at 500, 000 to 600, 000 cells per 75 square centimeter flask. This 100 x photo micrograph shows a GS adult hippocampal neuro stem cells after three days of growth in A-G-S-N-S-C.
Basal media in polyol ornithine coated flasks as FBS and RH FGF basic are removed. The A GS neuro stem cells will differentiate to about half neurons and half astrocytes. The neurons will be T tuj one positive within 14 to 21 days as illustrated by this fluorescent micrograph.
Here, the cells were fixed with 4%paraform, aldehyde, and neurons identified by green. Using TJ one primary antibody and LOR 5 68 secondary antibody, all nuclei were stained blue by the addition of hooks die. The ratio of neurons to total cells will range between 40 to 60%dependent on the age of the culture.
This graph shows the number of neurons and the neuron ratios have differentiated a GS neuro stem cells at 16, 19, and 21 days after inoculation. For differentiation, neurons may be utilized for experimentation from 12 days post seating through at least 21 days post seating. After watching this video, you should have a good understanding of how to prepare arctic ground squirrel neurons for use in ischemia experiments.
