The aim of this video protocol is to isolate and expand neuro stem cells from the embryonic mouse brain using the neuros sphere assay. The procedure includes harvesting E 14 mouse embryos followed by brain micro dissection to harvest the ganglionic eminences dissociation of the harvested tissue to gain a single cell suspension, and finally plating the Cells in neuros sphere culture. Hi I, I'm Hassan Ali from the laboratory of Dr.Brent Frennels at the University of Florida Mite Brain Institute.
Today I'm gonna show you how to establish a neuros sphere culture from the embryonic mouse brain using the neuros sphere assay. We routinely use this assay in our lab to isolate and expand neural stem cells. Okay, Let's get started.
Anesthetize A time Mated pregnant mouse. On day 14 of gestation, perform cervical dislocation and rinse the abdomen with 70%ethanol. Grasp the skin over the abdomen using large forceps, and then cut the skin and the underlying fascia with large scissors.
To expose the abdominal cavity and the uterine horns, remove the uterine horns containing the embryos and transfer them into a 50 ml conical tube containing cold him. After transferring the uterine tissues to the PC two hood, remove the hem from the 50 ml tube and rinse the tissues with enough volume of fresh cold hem to remove contaminants like blood and hair, perform two to three washes like this. Transfer the uterine tissues to a 10 centimeter Petri dish containing cold hem.
Open the uterine horns using small curved forceps and scissors and transfer the embryos to a new dish containing cold hem. Repeat this procedure until all of the embryos have been harvested. Then place the embryos under the dissecting microscope.
Separate the heads of the embryos at the cervical spinal cord level and transfer them to another Petri dish containing cold hem. Hold the head with fine curved forceps using micro scissors. First, make a horizontal cut above the nose and then continue to cut in the midline from the forehead towards the back of the head.
Make sure to cut through the skin and the skull and not to damage the underlying brain. Remove the brain from the skull by pushing the edges of the cut section in a backward motion using the curved forceps. Continue this procedure until all brains are removed from the skulls.
Hold the brain steady using the curved forceps so that the dorsal side is facing upwards, and then using micro scissors cut through the cortex of each hemisphere from the olfactory bulbs to the back of the hemisphere. To expose the ganglionic eminences Holding the brain in place, Dissect out the whole ganglionic eminence with 45 degree angled forceps and transfer it to another Petri dish. The ganglionic eminence is clearly shown here with its medial and lateral parts.
Alternatively, you can dissect the ganglionic eminences by removing the surrounding tissues Using fine forceps. You can also cut the Whole eminence into a medial and lateral half. If you're interested in growing cells from either of These regions.
Repeat this procedure until all the brains have been micros dissected. Use one milliliter of neural Stem cell medium to collect tissue pieces and transfer them to a 15 milliliter centrifuge tube. Allow the tissue clumps to settle in the 15 milliliter tube in order not to leave any tissue behind.
Remove some of the supernatant medium and wash the base of the Petri dish to transfer any additional tissue to the 15 ml tube. Placing the pipette tip against the bottom of the tube. Pipette the suspension up and down three to four times to break up the tissue achieving a single cell suspension.
Then let the suspension settle for one minute so that the non associated clumps precipitate transfer almost the entire cell suspension to another 15 ml tube. Then add one milliliter of neuro stem cell medium to the remaining clumps and dissociate them To single cells. Pull the contents of the tubes.
Then centrifuge the suspension at 700 RPM or 110 G for five minutes. Remove the supernatant and resuspend the cells in one milliliter Of complete neuro stem cell medium. Take 10 microliters Of the cell suspension and mix well with 90 microliters of trian blue.
Then take 10 microliters of this suspension and transfer it to a hemo cytometer. Perform a cell count. Transfer a total of 20 mls of complete neural stem cell medium to a 50 ml conical two in order to plate one T 80 flask.
Then mix the cell suspension and add an appropriate amount of cells for a total density of 200, 000 cells per milliliter or 4 million cells per T 80 flask. Add EGF at a final concentration of 20 nanograms per milliliter. Mix the cells that are in complete neural stem cell medium with growth factor and transfer the cell suspension into a T 80 flasks.
Label the flask and look at the cells under the microscope to ensure you have a single cell suspension without tissue clumps. Transfer the flask to an incubator at 37 degrees Celsius with 5%CO2 for five to seven days. After five to seven days, evaluate the flask under the microscope.
By this time, neurospheres have formed and should be between 150 to 200 microns in diameter and are ready to be subculture. Collect the contents of the flask in an appropriate size sterile tissue culture tube, centrifuge the tube and discard the supernatant. Resuspend the spheres in one middle milliliter of prewarm trypsin and place the tube in a 37 degree water bath for two minutes.
Then add an equal volume of trypsin inhibitor and mix well but gently to stop the trypsin activity Centrifuge again and remove the snat. Then resuspend the cells in one Milliliter of complete neural stem cell medium and perform a cell count as mentioned before. In order to plate one T 1 75 flask, transfer a total of 40 mls of complete neural stem cell medium to a 50 ml conical tube.
Then mix the cells and add an appropriate amount of cells for a total density of 50, 000 cells per M or 2 million cells per T 1 75 flask. Then add EGF at a final concentration Of 20 nanograms per mil. Mix the cells that are in complete neural stem Cell medium with growth factor and transfer the cell suspension into a T 1 75 flask.
Label the flask and transfer it to an incubator for five to seven days. This is an example of primary E 14 Neural stem cell culture. Three days after plating, the arrows show proliferating clusters of neural stem cells.
You can also see cells that are attached to the substrate and are differentiated. This is an example of primary E 14 neuros stem cell culture seven days after plating. As you can see, there are spheres of various sizes.
There are also cells that are differentiated. The amount of debris is much lower than the primary adult neuros sphere.Cultures. Also, the spheres are not compacted and have micro spikes around the periphery indicating that the spheres are healthy and viable.
The sphere should not be allowed to grow too large as they might become dark in color due to the cell death at the center. Large spheres might Also attach to the substrate and differentiate. This is an example of Passage one E 14 neurospheres five days after plating.
Here again, you can see the micro Spikes at the periphery of the spheres. We've just shown you how To grow neurospheres from E 14 mouse ganglionic eminences following the same procedures described here. You can also grow neurospheres from other reasons of the brain like cortex and mesencephalon.
Thank you for watching and good luck with your neuros sphere cultures.
