Isolating and Culturing Cells from Diffuse Intrinsic Pontine Glioma

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.

In this procedure, centrifuge the conical tube containing the remaining tissue fragments for five minutes. After that, remove the supernatant and add the pre-warmed enzymatic digestion solution such that there is 5 milliliters of digestion solution for every 1 milliliter of tissue. Then, seal the conical tube lid with laboratory film and incubate the reaction on a rotator at 37 degrees Celsius for 30 minutes.

After incubation, triturate the samples gently. Using a 10-milliliter serological pipette, pipette the sample up and down for 6 to 8 times and avoid generating excessive air bubbles. Next, add a 1000-microliter pipette tip to the end of the pipette and triturate the sample for an additional 6 to 8 times.

Allow any remaining chunks to settle to the bottom of the tube. Then, remove and filter the supernatant with the cell still suspended through a 100-micron filter into a new 50-milliliter conical tube labeled enzymatic dissociation, and store it on ice. Following that, centrifuge the enzymatic dissociation tube for five minutes and continue to sucrose gradient centrifugation. If the sample is still suspended in solution, centrifuge it for five minutes.

Next, remove the supernatant, and resuspend the tissue in 20 milliliters of cold HBSS without calcium and magnesium. Then, bring the volume up to 25 milliliters with cold HBSS. Slowly, add 25 milliliters of 1.8 molar sucrose solution and invert the tube to mix. This results in a 0.9 molar sucrose gradient.

Subsequently, centrifuge the sample with no break for 10 minutes. Carefully aspirate the myelin debris and as much sucrose solution as possible. Then, wash the sample by adding 30 milliliters of cold HBSS without calcium and magnesium, and mixing gently. After that, centrifuge it for five minutes.

In this procedure, remove the wash supernatant. Add 5 milliliters of ACK lysis buffer and gently resuspend the cell pellet, swirling the tube for one minute at room temperature. Then, quench the lysis by adding 30 milliliters of cold HBSS without calcium and magnesium.

Subsequently, centrifuge it for five minutes. Resuspend the final cell pellet in 10 to 15 milliliters of warm complete TSM with growth factors, and quantify the viable cell density on a hemocytometer using trypan blue exclusion. Afterward, transfer the final cell suspension to a new T75 culture flask. Add additional growth factors every other day to maintain the overall growth factor levels and monitor for the development of tumor cell neurospheres.