Isolation of Neural Stem Progenitor Cells from the Spinal Cord Periventricular Region of an Adult Rat
Transcrição
Begin with an adult rat's spinal cord periventricular tissue, a region rich in neural stem/progenitor cells.
Transfer the tissue to a solution containing papain and DNase.
Papain, a proteolytic enzyme, cleaves the tissue's extracellular matrix, while DNase degrades contaminating DNA released from damaged cells.
Mechanically dissociate the tissue to obtain a single-cell suspension.
Transfer the suspension to a tube and centrifuge it. Remove the supernatant. Resuspend the cells in a solution containing albumin protein and a protease inhibitor.
Albumin and the inhibitor deactivate papain.
Layer the cell suspension over a solution containing albumin and a protease inhibitor.
The difference in densities between the layers forms a discontinuous density gradient.
Centrifuge to settle the intact cells, while cell membrane fragments remain at the interface.
Discard the supernatant. Resuspend the cells in growth media and plate them.
The growth factors selectively promote the proliferation of the neural stem/progenitor cells and their aggregation into three-dimensional neurospheres.
