Our research is focused on studying how the neural stem cell population is regulated in the adult mammalian brain. Understanding the intrinsic and extrinsic molecular mechanisms that regulate neural stem cells within the neurogenic niches is important to comprehend the biology and to develop future potential therapeutic applications. To study neurosensory behavior, both in-vivo and in-vitro approaches are widely used, in-vitro neural stem cell cultures of their controlled environment and the possibility to easily manipulate and monitor this population of cells.
Techniques for gene expression perturbation in vitro are a versatile approach to study molecular mechanisms while learning cell biology. However, an efficient and reproducible method for overexpressing and knocking down candidate genes in neural stem cells is still challenging in the field. Traditional transfection methods for gene delivery have proven effective in central nervous system cells.
Moreover, these methods affect cell viability and functionality. Thus, refinement of alternative approaches is crucial to manipulate gene expression in neural stem cells. With this protocol, we present an improved nucleofection system that achieves high efficiency of gene delivery in neural stem cell cultures from the adult murine subventricular zone, along with survival rates higher than the 80%To begin the extraction of the brain from the properly euthanized mouse, after separating the head from the rest of the body, expose the brain by initially cutting the skull along the sagittal suture.
With the help of fine tweezers, remove the bones. Be careful not to damage the brain tissue beneath the skull during this step. Using a spatula, carefully extract the brain from the skull.
Place it into a 12-well plate containing cold Dulbecco's phosphate-buffered saline, or, DPBS. Next, transfer the entire brain onto the silicon pad where the dissection will take place. Use a scalpel to remove the olfactory bulb located at the rostral end of the brain and the cerebellum situated in the caudle part while retaining the central portion of the brain containing both lateral ventricles.
Then divide the brain along the longitudinal fissure into two hemispheres before proceeding with the dissection of both hemispheres separately. Working with one hemisphere, reorient it so that the medial area faces upward. Open the brain along the line of the corpus callosum, separating the cortex and striatum from the hippocampus, septum, and diencephalon, thereby exposing the lateral ventricles.
Remove the hippocampus, septum, and diencephalon following the ventral limit of the ventricles. Then remove the tissue beyond the rostral and caudle ends of the subventricular zone or, SVZ, and the cortex following the corpus callosum. Tilt the tissue so that the SVZ faces sideways.
Remove the striatal tissue beneath the SVZ to obtain a thin piece of tissue containing the neural stem cells. Store both dissected SVZs from the mouse in a 12-well plate containing cold DPBS till tissue processing. To isolate neural stem cells or, NSCs, from the dissected murine subventricular zones or, SVZs, cut each SVZ into four to five small pieces to facilitate the disaggregation of the tissue.
Using a sterile plastic Pasteur pipette, transfer the chopped SVZs to a 15-milliliter centrifuge tube while ensuring no fragments are left in the plate. Let the tissue sediment at the bottom of the tube before removing the remaining DPBS. Add 500 microliters of filtered papain-containing enzymatic mix for the two SVZs per brain, and incubate the tube in a water bath at 37 degrees Celsius for 30 minutes.
After incubation, add five milliliters of control medium pre-warmed at 37 degrees Celsius to each sample. Centrifuge the sample at 300g for five minutes. Carefully remove the supernatant using a micropipette or a vacuum pump.
Using a fire-polished Pasteur glass pipette, add one milliliter of control medium to the pellet. Mechanically disaggregate the pellet carefully by pipetting up and down 10 to 20 times until a homogeneous cell suspension is obtained. Then add four milliliters of control medium and mix by inversion to wash the cells.
After centrifuging at 300g for five minutes, homogeneously resuspend the resulting pellet in one milliliter of complete medium by pipetting up and down 10 times. After diluting one part of an aliquot of the cell suspension with one part of trypan blue, count the cells using a Neubauer chamber under the microscope. Ensure cellular disaggregation at the single-cell level before seeding the cells equally in eight wells of a 48-well plate in a final volume of 500 microliters of complete medium.
Incubate the cells for five to seven days to let the primary neurospheres form. To perform passage of neurospheres obtained from neural stem cells isolated from murine subventricular zone, collect the medium containing the neurospheres from the multi-well plates and transfer them to a 15-milliliter centrifuge tube. Centrifuge the neurospheres for five minutes at 300g.
After removing the supernatant, add 200 microliters of enzymatic solution to the pellet and gently tap the bottom of the tube to dislodge it. Incubate for 10 minutes at room temperature to facilitate the neurosphere dissociation. Then add one milliliter of control medium to halt the enzymatic reaction.
Mechanically dissociate neurospheres with a P-1000 micropipette by pipetting up and down 10 to 20 times. Add an additional volume of four milliliters of control medium and mix by inversion to wash the cells. Centrifuge the cells for five minutes at 300g before removing the supernatant.
Add one milliliter of complete medium and resuspend the pellet to homogenize the cellular suspension by pipetting up and down 10 times. After diluting one part of an aliquot of the cell suspension with one part of trypan blue, count the cell suspension using a Neubauer chamber. To expand the culture, seed cells at a density of 10, 000 cells per square centimeter using complete medium in an appropriate culture plate or flask.
Using the recommended kit, proceed to prepare 95 microliters of the nucleofection solution in a 1.5-milliliter tube for each desired nucleofection condition. Also, prepare a T25 flask filled with four milliliters of pre-warmed complete medium per condition and keep it in the incubator until use. To perform nucleofection, centrifuge the desired number of cells for five minutes at 300g before removing the supernatant.
Add one milliliter of DPBS and resuspend the pellet homogeneously by pipetting up and down 10 times. After repeating the centrifugation, resuspend the pellet in 95 microliters of nucleofection solution. Combine the 95 microliters of cells with a premixed solution containing five microliters of each plasmid selected for nucleofection.
After transferring this solution to a cuvette, place it in the Nucleofector Device to nucleofect the cells with the plasmids using the optimized neural stem cell program of the Nucleofector system. After completing the electroporation, using the Pasteur pipette provided in the kit, carefully introduce warm complete medium into the cuvette. Transfer the contents of the cuvette to the previously prepared T25 flask containing pre-warmed complete medium.
Incubate the nucleofected cells in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide for three to five days and visualize the nucleofected neurospheres.
