Isolating and Culturing Cells from Diffuse Intrinsic Pontine Glioma
Transcription
Begin with brain stem tissue fragments containing diffuse intrinsic pontine glioma, a tumor derived from glial progenitor cells in a myelinated nerve-rich environment.
Treat with proteolytic enzymes to degrade the tissue’s extracellular matrix.
After incubation, repeatedly pipette the contents to facilitate cell dissociation and degradation of the myelin sheath.
Filter and centrifuge to collect the cells. Remove the supernatant and resuspend them in a cold buffer.
Add a sucrose solution for density gradient separation and mix thoroughly.
Next, centrifuge to separate the heavier cells from the lighter myelin debris.
Remove the myelin layer and add a RBC lysis buffer.
Add a cold buffer to stop lysis action. Centrifuge and remove the supernatant to eliminate the lysed RBCs.
Resuspend the cells in a warm neurobasal medium and culture them in a non-coated flask.
The absence of neurotropic factors causes neuronal death, while the glial progenitor cells utilize nutrients and growth factors, leading to proliferation. Add fresh medium to maintain growth factor levels.
Over time, these cells spontaneously aggregate into cell clusters, forming free-floating neurospheres.
