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 c