The goal of this protocol is the isolation and expansion of human glioblastoma multiform tumor cells using the neurospheres asset. This is achieved by first mincing the harvested tumor tissue, followed by ization mechanical dissociation, and finally plating the resulting cells To grow in neuros sphere culture. In this Video, we are going to show how to isolate and expand stem-like cells from human glioblastoma tumor tissue using the neuros sphere culture method.
This method can also be used to culture cells from other brain tumors as well as solid tumors such as breast, prostate, liver, and lung. Okay, let's get started. After receiving the resected GBM tumor Tissue from surgery, the medium is removed.
The tumor is washed two or three times using sterile PBS to remove contaminants Such as blood and debris. After washing, place the resected tumor into sterile petri Dish. First, cut the tumor into smaller portions, then transfer a portion of tumor tissue into a separate Petri dish, mince the tissue using a number 10 scalpel into tiny pieces.
This will increase surface area available for enzymatic dissociation. Mincing can take one to three minutes depending on the size of the tumor. Now add three to five ml of prewarm trypsin to the mince tissue and transfer it to a 15 ml sterile falcon tube.
Place the 15 Ml tube in 37 degree water bath for 10 to 15 minutes. Shake the tube every five minutes. To ensure better tissue digestion, add an equal volume of trypsin inhibitor to stop the reaction.
Trypsin in activation is insured by pipetting up and down several times. Centrifuge the suspension at 800 RRP M or 110 G for five Minutes. Remove the supernatant, then resuspend the pallet in One ml of neuros stem cell basal media, placing the tip against the bottom of the tube.
Pipette up and down three to seven times to break up clumps until you achieve a milky suspension over pipetting might result in cell death. Place a 40 micron filter on a 50 ml tube. Now add 10 to 15 ml of basal medium to cell suspension and mix it gently pass the cell suspension through the 40 micron filter so as to remove debris in unassociated clumps.
PT the cells And vacuum off the supernat. Resuspend the Cells in one to two mls of complete medium. Then gently pipette up and down to homogenize the suspension.
Mix 10 microliter of cell suspension in 90 microliter of Trian blue. Mix the suspension well and transfer 10 microliter to hemo.Cytometer. Perform a cell count as shown here.
A single cell suspension is required with no clumps to have an accurate cell count. Do not include trian blue positive cells. When counting to plate the cells in a T 80 flask.
Add 20 ml of complete neuro stem cell media to a 50 ml tube. Add appropriate amount of cell suspension to reach a density of 200, 000 cells per ml. Now add EGF to reach a final concentration Of 20 nanogram per ml.
Add 0.2%heparin at concentration of one microliter per ml. Finally, add BFGF to reach a Final concentration of 10 nanogram per ml. Mix the Cells in complete media supplemented with growth factors.
Well then transfer the suspension to a T 80 label. The flask then transfer to incubator at 37 degrees Celsius and 5%CO2 for seven to 10 days. After eight days in culture, the spheres are fully grown and ready to be passaged.
Collect the contents of the flask and transfer to a 50 ml two centrifuge. The cells at 800 RPM for five minutes. Vacuum off the supernat, then resuspend the pallet in one ml of prewarm trypsin.
Incubate the cells in water bath at 37 degrees Celsius for two minutes. Add an equal amount of trypsin inhibitor and mix thoroughly to ensure an activation. Then pellet the cells.
Remove the supernat and resuspend the cells in one ml of complete medium pipet up and down gently to homogenize the cell suspension. Perform a cell count as described earlier to plate the cells in a T 80 flask at 20 ml of complete medium in a 50 ml tube. Add an appropriate amount of cell suspension to reach a density of 50, 000 cells per ml.
Now add Growth factors including E-G-F-B-F-G-F, and heparin at the concentration described earlier. Mix the resulting cell suspension gently, then transfer the suspension to a T 80 and place in the incubator for seven to 10 days. Here's an example of primary GBM Culture.
After four days at this stage, you can see the cells are proliferating and beginning to form spheres. You should not observe large spherical clusters of cells at this stage. This would've resulted from inappropriate tissue preparation.
After being in culture of seven to 10 days, spheres of varying size have formed when the spheres have reached an average diameter of 150 to 200 microns, They're ready to be passaged. This is an example of passage GBM culture after eight days. Again, you can see the spheres of varying sizes, healthy spheres, exhibit micro spikes on their periphery.
Preparing a single cell Suspension is a very important issue in any sphere cultures. Otherwise, you might end up observing cell aggregates even on the day after plating cells. These aggregates be assumed as spheres, but it should be noted that it takes at least a week for perforating cells to form true spheres.
The amount of debris in primary tumor sphere culture varies depending on many factors such as source of tissue and that how precise the tissue has been removed and also the tissue processing technique. We hope you find this video useful and wish you good luck with your experiments.
