Generation and Differentiation of Primary Neurospheres from Zebrafish Neural Stem Cells

Begin with a multiwell plate containing a growth medium enriched with growth factors.

Seed a single-cell suspension of zebrafish neural stem cells and incubate. 

Initially, the stem cells utilize the growth factors and proliferate.

Remove debris and add the fresh medium to promote continuous cell proliferation and spontaneous aggregation to form free-floating primary neurospheres.

Next, transfer the neurospheres to a fresh well containing the growth medium.

Incubate under the same conditions to induce a progressive expansion of the neurospheres.

Pipette repeatedly to mechanically dissociate the neurospheres into small clusters.

Next, transfer them onto an extracellular matrix coated well, facilitating their adhesion. 

Replace the growth medium with a growth factor-free differentiation medium containing insulin.

The absence of growth factors and the presence of insulin stimulate cell differentiation into glial cells and neurons with neuronal projections.

To prepare for neurosphere generation, fill each well of a 24-well plate with 300 microliters of fresh Z-condition medium. Then, add 200 microliters of the cell suspension to each well, seeding them at a density of approximately 500 cells per microliter. Afterwards, incubate the plate at 30 degrees Celsius in 5% carbon dioxide.

After one day of culture, view the cells in the 24-well plate under a microscope. Expect to observe single-cell suspensions at this stage. If any debris is seen to have collected in the center of a well, remove it by pipetting out approximately 100 microliters of medium, and replace this liquid by adding 100 microliters of fresh Z-condition medium. Once all debris has been removed, incubate the plate under the conditions described previously.

After an additional day of culture, transfer 250 microliters of cell suspension from a single well into a new, empty well. Repeat this step for all 24 wells. Then, add 250 microliters of fresh Z-condition medium to each well, and homogenize the suspensions by gently pipetting up and down. Incubate the plates again under the same conditions and repeat this expansion procedure after an additional day of culture. Expect to observe a progressive increase in the size of neurospheres on the third and fourth days of this period.

To begin passaging, after four days, remove 250 microliters of medium, but no neurospheres, from each well. Then use a 1-milliliter pipette to mechanically dissociate the neurospheres in the remaining medium volume. Proceed to pool the cell suspension from each dissociated well. Then, count the cells with a hemocytometer, and distribute 250 microliters of primary culture supernatant containing 800 cells per microliter into each well of a new 24-well plate. Continue by adding 250 microliters of Z-condition medium to each well and then incubating the plate as previously described.