The overall goal of this adult neural cell culture system is to isolate adult neural stem cells from the subcallosal zone and expand the cells in nonadherent and adherent culture systems. This message can help answer key questions in the adult neural stem cell field such as isolation and culturization of neural stem cells from small brain lesions such as subcallosal zone. The main advantage of this technique is the isolation of adult neural stem cells from the subcallosal zone.
It's simple, but precise. To begin this procedure, autoclave the brain matrix, a double edged razor blade, and forceps. Next, prepare 50 milliliters of cold PBS for washing the mouse brain.
Then, set up a dissection microscope and prepare the surgical tools required for brain dissection and SCZ isolation. Prepare F-12 DMEM medium with 2%B-27 and 1%N-2 supplements and 1%penicillin-streptomycin. Then, prepare the digestion buffer for tissue digestion.
After that, prepare 0.01%Poly-L-Ornithine and laminin. Following that, coat six small plates for the maintenance of SCZ adult neural stem cells as a monolayer, or 18 millimeter cover slips for immunostaining. Then, incubate them with Poly-L-Ornithine overnight at four degrees Celsius.
Afterwards, wash them three times with DH20 and allow them to dry after the last wash. After that, incubate the plates with laminin overnight at four degrees Celsius. Once dissected, transfer the mouse brain to cold PBS buffer and rinse it twice to remove excess blood.
Next, place the brain in the brain matrix on ice and make coronal cuts to obtain one millimeter thick slices. Subsequently, transfer the posterior brain slices containing SCZ regions to cold PBS. Under a dissecting microscope with low magnification, micro-dissect the SCZ from the white matter area of the cortex and the hippocampus with a bent 30 gauge needle.
Then, remove the cortex regions above the SCZ and place the dissected SCZ into cold PBS in a 35 millimeter Petri dish on ice. The region of subcallosal zone can be dissected precisely from the whole tissue by using brain metrics and bent 30 gauge needle. Following that, remove the PBS and then immediately chop the dissected tissues into small pieces.
Resuspend them with one milliliter of digestion buffer. Afterward, transfer the sample to a 15 milliliter tube containing two milliliters of digestion buffer. Then, incubate it for 30 minutes in a 37 degree Celsius water bath.
In this step, tap the tube mildly to dissociate the digested tissue. And then centrifuge the tube at 145 x g for five minutes. After five minutes, discard the supernatant, resuspend the digested tissue with 1 milliliter of pre-warmed N-2 medium to wash out the digestion buffer and gently pipet the sample solution a maximum of five times.
Next, centrifuge the sample again at 145 x g for five minutes. After discarding the supernatant, resuspend the cell pellet in one milliliter of N-2 medium. Place one milliliter of N-2 medium in a non-coated six-well plate.
And add one milliliter of the suspended cells to make a final volume of two milliliters. Next, add 20 nanograms per milliliter EGF and 20 nanograms per milliliter bFGF into each well. Gently shake the six-well culture dish by hand, to mix the added growth factors with the plated cells.
Then, keep the six-well plate in a 37 degree Celsius and 5%carbon dioxide incubator. Add 20 nanograms per milliliter EGF and 20 nanograms per milliliter bFGF to each well every day for eight days. Every third day, add 200 microliters of N-2 media to maintain approximately two milliliters of the medium.
Here is an image of the neurospheres after eight days. In this procedure, gather the neurospheres and transfer them into a new 15 milliliter conical tube. Centrifuge the tube at 145 x g for five minutes.
Subsequently, discard the supernatant and resuspend the neurospheres with 0.5 milliliters of dissociation buffer. Incubate them with 0.5 milliliters of dissociation buffer for five minutes in a 37 degree Celsius water bath, to dissociate the neurospheres into single cells. Primary neurospheres can be dissociated into single cells and maintained over several passages as a neurosphere or monolayer culture.
Gently pipet the sample solution up and down less than five times and centrifuge the tube at 145 x g for five minutes. Subsequently, discard the supernatant and resuspend the neurospheres with one milliliter of N-2 medium. To count the cells, make 10 microliters of a cell suspension and 0.4%Trypan blue solution mixture, and count the number of live cells on a hemocytometer.
After coating a six-well plate with PLO/Laminin, plate the cells at 2.5 x 10 to the fifth cells per milliliter, with two milliliters of N-2 medium per well. Maintain the SCZ adult neural stem cells with a daily treatment of growth factors for five days before passaging them. To differentiate SCZ adult neural stem cells, plate adult neural stem cells onto the PLO/Laminin coated 18 millimeter cover slips with 20 nanograms per milliliter growth factors.
The next day, when the cells are firmly attached to the cover slips, replace the growth medium with N-2 to remove the growth factors. After six days, wash the differentiated cells with one milliliter of PBS to remove the cell debris and fix them for immunostaining. Shown here are the representative images of SCZ adult neural stem cells in the monolayer culture system, which shows the increase of the number of SCZ adult neural stem cells after three days in vitro.
Here are the representative images of SCZ adult neural stem cell proliferation using the BrdU essay. Cells maintained in the monolayer culture system were not stained with markers for type C transient amplifying cells and type A neuroblasts. To confirm the differentiation capacity of SCZ adult neural stem cells, growth factors were removed from the culture media.
After six days, cells were immunostained with markers for neurons, astrocytes, and oligodendrocytes. Once mastered, this technique can be done in 1.5 hours if it is performed properly. While attempting this procedure, it's important to remember to maintain the dissected tissue on ice and perform the procedure with precision.
Following this procedure, other methods like effects can be performed in order to answer additional questions, like analyzing the intrinsic properties of subcallosal zone derived adult neural stem cells. After its development, this technique paved the way for researchers in the field of other neural stem cells to explore their potential for regeneration in brain damages. After watching this video, you should have a good understanding of how to culture other neural stem cells precisely from the subcallosal zone and maintain them in vitro.
