The overall goal of this procedure is to study brain tumor initiating cells in vitro. This is accomplished by first mechanically and enzymatically dissociating primary human tumors and then culturing them in stem cell serum free culture conditions. Next sphere formation is monitored over three to five days.
Tumor spheres are then enzymatically digested again, and their surface markers are evaluated by flow cytometry. The final step is to sort the desired cells into 96 well plates or PL grid slides for further characterization. Ultimately, the self-renew ability and differential gene expression of the sorted population can be assessed by limiting dilution analysis and single cell R-T-P-C-R.
The main advantage of this technique over existing methods, such as bulk tumor analysis, is that it prospectively isolates the brain tumor initiating cell or cancer stem cell populations. We first had the idea for this method when we applied the culture conditions originally used for normal neural stem cells to a variety of human brain tumors, and found that this culture method specifically selected for stem cell-like populations demonstrating the procedure will be postdoctoral research associate CHITRA AL graduate students, bran Van Manen and Sarah Nolte, and our flow cytometry technician Nicole McFarland, all from our laboratory Begin by adding 200 microliters of thaw liase to 15 milliliters of artificial cerebral spinal fluid or A CSF, and then placing the tube into a 37 degree Celsius water bath. Bring ammonium chloride solution to room temperature at this time as well.
Then at five milliliters of A CSF to a specimen container of brain tumor tissue, swirl the container to rinse and then pipe it off the fluid to remove the red blood cells. Now transfer the tissue to a sterile 100 millimeter Petri dish and use fine scissors and forceps to disaggregate the tissue to a slurry consistency. Then collect the sample using a 10 milliliter regular pipette and forceps and transfer the tissue fragments into the tube containing the prewarm A CSF.
With liase, place the tube on an incubator shaker at 30 RPM and 37 degrees Celsius. And then after 15 minutes, filter the tissue lysate through a seven D micrometer cell strainer into a 50 milliliter conical tube. Spin the filtrate down for five minutes at 280 GS at room temperature.
Then carefully remove the snat and evaluate the size and color of the resulting pellet. Next, resuspend the pellet in one milliliter of PBS and then add an appropriate amount of ammonium chloride solution based on the pellet size and the red cell contamination. Then after a five minute incubation at room temperature, spin down the cells after a second wash in PBS resuspend the pellet in five milliliters of neuro stem cell complete medium, and then transfer the cell suspension to an ultra-low binding 60 millimeter tissue culture plate for the first few days.
Top off the cultures with one to two milliliters of media as needed, changing the media only when the color becomes slightly yellow. Begin by transferring the tumor spheal culture to a 15 milliliter conical tube. Add two to three milliliters of sterile PBS to rinse the plate thoroughly and then add the wash to the tumor sphe tube.
After spinning down the cells, remove the snat and resuspend the pellet in one to two milliliters of sterile PBS and then add 10 microliters of liase. One challenge of this technique is to maintain the viability of the solid tumor stem cells during processing. Any manipulation of the cells during culturing and sation requires a gentle touch.
Next, incubate the tube in a 37 degrees Celsius water bath. After three minutes, remove and visually evaluate the suspension. If multiple clumps are seen, use a 1000 microliter pipette tip to gently iterate the cell cluster.
Now wash the cells in five milliliters of sterile PBS and then after removing the S supernatant, resuspend the pellet in 500 to 1000 microliters of sterile PBS plus two millimolar EDTA. Finally, after assessing the cell number and viability using Trian blue, adjust the cell count to 1 million cells per milliliter in PBS plus EDTA. Begin by transferring 100 microliters of the cell suspension per test to individual 12 by 75 millimeter flow tubes.
After adding the appropriate antibodies, incubate the samples on ice for 30 minutes, then wash away any unbound antibody in two milliliters of PBS plus EDTA per tube. Now decant the wash solution, blot the tubes and then resuspend the cell pellets in 300 more microliters of PBS plus EDTA. Then add 10 microliters of the seven A a D viability dye to each tube.
After incubating the tubes for at least 15 minutes on ice, the cells are ready for flow cytometric analysis. After using the negative control sample to adjust the forward and side scatter parameters, draw a region around the cells of interest similar to the one shown in this representative top plot. Adjust the voltage on the fluorescence detectors utilized to place the negative cells within the first two decades as necessary.
Next, use the viability dye seven A a d to create a second region for the exclusion of dead cells similar to the region shown in this representative top plot. Then apply both the scatter and live cell gates to all fluorescence plots and use the single stained controls to subtract any spectral overlap. Finally, run the samples.
Begin the limiting dilution assay by sorting the dissociated cells at varying dilution to a single cell per well into individual wells of a 96 well plate containing 200 microliters of NSC media neurospheres usually form over a period of seven days and the rate of the subsequent sphere formation over the past seven days in culture can be calculated for single cell R-T-P-C-R. After sorting single cells from the populations of interest onto the reaction sites of an amply grid slide, add one microliter of master mix to the center of each reaction site, and then immediately coat each site with five microliters of ceiling solution. Then using an amply speed slide cycler, incubate the slide for three temperature cycles.
After the reverse transcription, add three microliters of DN a's rase free water to each reaction site. Then transfer the entire mixture from each reaction site to individual PCR tubes. Finally, for real-time quantification, run the samples on a BioRad cycler.
This first figure shows a magnetic resonance imaging scan of a representative patient with glioblastoma. The dissociated tumor cells can be labeled with the neural surface stem cell markers, CD 1 33 and CD 15, and analyzed by flow cytometry as shown in this representative dot plot. Here, the amplification plot for representative single cell R-T-P-C-R reaction is shown.
The CT values obtained from such a plot can be used to calculate the relative expression levels of the genes of interest. After watching this video, you should have a good understanding of how to enzymatically dissociate and culture primary brain tumor tissue to enrich for stem cell populations. You'll also know how to study the self-renewal ability and differential gene expression of sorted stem cell populations by performing limiting dilution analysis or single cell R-T-P-C-R.
