The aim of this video protocol is establishing a source and efficient protocol for Embryonic Mouse Neural Stem Cell Isolation and Culture. Hello, I'm Maryam Sadeghi-Zadeh. I'm a Master student of Cellular and Molecular Biology in Royan Institute.
Today me and my colleague want to show you how to harvest neural stem cell from 13 embryo mouse ganglionic eminence. Hello, I am Bahareh Alizadeh-Shoorjestan. I'm a Master student for Cellular and Molecular Biology in the Stem Cell Department of Royan Institute for Biotechnology.
Hello, I am Reza Dehghani-Varnamkhasti. Also I'm studying Cellular and Molecular Biology in Royan Institute. Surgical procedures include harvesting each 13 mouse embryos from uterus.
Cutting the front of fontanel of embryo with a bent needle tip extracting the brain from the skull. Micro-dissection of isolated brain to harvest the ganglionic eminence. Dissociation of the harvested tissue in a neural stem cell medium to gain a single cell suspension.
And finally, plating cells in a suspension culture to generate nerve cells. Clean and disinfectant the skin of abdomen area by spraying with 70%ethanol. Control the skin upward abdomen and then cut the skin and underlined facial with scissors to uncover abdominal cavity and uterine horns.
Remove the uterine horns containing the embryos by scissors and transfer them into a 50 ml conical tube containing 25 ml cold HEPES MEM buffer. Place the conical tube under the laminal flow hood. Open the cap under the hood.
Bring the uterine horns out from the tube, and rinse three times with enough volume of the fresh cold HEPES MEM buffer to eliminate debris and blood. Transfer the uterine tissue to a 10 cm petri dish containing 7 to 10 ml cold HEPES MEM buffer. Open the uterine horns using fine scissors and transfer embryos to a new dish containing 7 to 10 ml of cold HEPES MEM buffer.
Now put the 10 cm dish with embryos under the dissecting microscope for micro-dissection operation. Bend the tip of a syringe needle by pushing its tip on a steel scalpel blade handle. Bend the tip of the needle until the tip is bent at an angle of 70 to 90 degrees.
Check the tip of the needle under the dissecting microscope to see the bend at the tip of the needle. Naturally embryos have a lateral position in this. Without changing their position, held the neck and body of the embryo with fine forceps and then insert the bent part of the needle deep into the front of the fontanel of the embryo head.
There would be a small bleeding or a blood clot there. Move the needle tip superficially while the bent part is inside the bulge toward forehead and then toward back of the head. Make sure to cut through the skin and the skull, and not to damage the underlying brain.
Push the skin with the needle tip laterally to uncover the brain. Insert the needle from lateral side of the skin to the beneath the frame from the lateral side of the skin to the area underneath the brain. And then remove the brain from this scalp by pushing it to come out from the dissected skull.
The ganglionic eminence is clearly shown in video with its medial and lateral parts. Held the brain steady using the care forceps and then using microscissors to cut the cortex of each hemisphere to expose the ganglionic eminence. Held the brain and place dissected ganglionic eminence into the 15 ml centrifuge tube containing neural stem cell media.
Repeat this procedure until all the brain have been micro-dissected. Cut dissected ganglionic eminence by placing the pipette tip against the bottom of the tube and pipette the suspension up and down for 2 5 times to break up the tissue to get a single cell suspension. Centrifuge the suspension at 110 g for 5 minutes.
Remove the supernatant and resuspend the cells in 1 ml of 0.05%tube in EDTA. Incubate the cell suspension in a 37 degree Celsius for 10 minutes. And then add an equivalent volume of soybean trypsin inhibitor to stop trypsin activity.
Pipette the cell suspension up and down to make sure that the trypsin has been totally inactivated. Centrifuge the cell suspension at 110 g for 5 minutes. Remove the supernatant and resuspend the cells in 1 ml of complete neural stem cell medium and count the number of cells.
Plate the cells at neural stem cell medium into the suitable size tissue culture flask. Transfer 5 ml of 2T25, 20 ml to T75, and 40 ml to T175 flasks. Neurospheres will appear after 3 days culture at 37 degree Celsius in a humidified incubator with 5%CO2.
To suck culture the neurospheres, transfer the medium with suspended neurospheres to the centrifuge to centrifuge at 110 g for 5 minutes, and then discard the supernatant. At 1 ml of 0.05%trypsin EDTA and incubate at 37 degree Celsius for 10 minutes. Then inactivate the trypsin using soyben tyrpsin inhibitor and pipette the neurospheres gently up and down to make them single cells.
Centrifuge the cell suspension at 110 g for 5 minutes. Discard the supernatant and resuspend the cells in 1 ml of neural stem cells medium. These cells are ready for next step culture or culture on laminate and fully coated surface for advanced experiments.
By cultivating one million primary neural stem cells after seven days the number of cells will increase to three to four millions. After six to seven days the spheres should measure between 150 to 200 micrometer in diameter and will be ready for sub culture on laminate poly ornithine coated dishes in absence of bFGF and EGF for neuronal differentiation. Flow cytometry assay results show that near to 95%of cells constituting the neurospheres were Nestin positive which is a known marker for neural stem cells.
Assays using beta tubulin III and ganglio fibrous protein antibodies reveal that by cultivating neural stem cells on coated surface around 94%shown neuronal phenotype and around 5%show glial phenotype. In just this short video a lab technique for rapid isolation of neural stem cell from 13 mouse embryo ganglionic eminence. I hope you will be success in your neural stem cell culture.
