Formation and Expansion of Neurospheres from a Hippocampal Tissue
Transcript
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.
