This article describes a method for isolating and culturing neural stem and progenitor cells from the postnatal mouse hippocampal dentate gyrus. The process involves enzymatic dissociation, cell culture, and the formation of neurospheres, which are essential for studying neural development and regeneration.
Begin with a postnatal mouse hippocampal dentate gyrus, a brain tissue rich in neural stem and progenitor cells with self-renewal capacity.
Add trypsin enzymes that dissociate the tissue's extracellular matrix, loosening the cells.
Next, remove the enzymes and wash the tissue repeatedly with a buffer.
Replace the buffer with a neuron culture medium enriched with growth factors.
Repeatedly pipette to dissociate the tissue and release the cells into the medium.
Seed the diluted cell suspension in an uncoated petri dish and incubate.
Stem and progenitor cells utilize nutrients and growth factors to proliferate.
Over time, they spontaneously aggregate, forming free-floating three-dimensional structures termed primary neurospheres with a heterogeneous cell population.
Collect these neurospheres and discard the supernatant. Resuspend the neurospheres in a neuron culture medium, and dissociate into a single-cell suspension.
Re-culture these cells in an uncoated petri dish to form secondary neurospheres with a relatively homogenous cell population.
To dissociate the SVZ and DG samples, add trypsin-EDTA 0.05% to a final concentration of 5% to 10% of trypsin-EDTA in HBSS to each tube for an approximately 15 minute incubation at 37 degrees Celsius.
The overuse of trypsin or a too-long incubation time can lead to increased cell death, negatively impacting cell growth.
And the tissue has clumped together, replace the enzyme solution for four consecutive washes with 1 milliliter of fresh, supplemented HBSS per wash. After the last wash, resuspend the digested tissues in 1 milliliter of serum-free medium, supplemented with 10 nanograms per milliliter of epidermal growth factor, and 5 nanograms per milliliter of basic fibroblast growth factor per tube.
Then, mechanically dissociate the tissues with gentle pipetting 7 to 10 times with a P1000 pipette, until a homogeneous cell solution has been obtained. To determine the density of the dissociated DG in SVZ cell, count the cells in each suspension on a hemocytometer.
To expand the cells into neurospheres, dilute the individual cell populations at a 2 x 104 cells per milliliter density, in serum-free medium, supplemented with growth factors, and seed 5 milliliters of the cell suspension into uncoated 60-millimeter diameter Petri dishes. Then, incubate the SVZ cells for six to eight days, and the DG cells for 10 to 12 days at 37 degrees Celsius for primary neurosphere formation.
When the majority of the neurospheres have a 150 to 200-micrometer diameter, harvest the cell suspension from the cultures, and collect the neurospheres by centrifugation. Re-suspend the neurosphere pellet with a mouse chemical dissociation kit according to the manufacturer's instructions.
Before collecting the cells with another centrifugation, replace the supernatant with 1 milliliter of serum-free medium supplemented with growth factors, and gently triturate the pellet about 10 times.
Count the dissociated neural cells as demonstrated, and reseed the cells at a 2 x 104 cells per milliliter density, in serum-free medium supplemented with growth factors into new 60-millimeter Petri dishes. Then, return the cells to the cell culture incubator for an additional 6 to 8, or 10 to 12 days as appropriate to obtain secondary neurospheres.
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