Formation and Expansion of Neurospheres from a Hippocampal Tissue
Transcription
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.
