This study investigates the tumor-killing potential of CAR T cells against glioblastoma using in vitro methods. The protocol involves culturing brain tumor spheroids and assessing the interaction between CAR T cells and tumor cells.
Cancer immunotherapy employs chimeric antigen receptor T cells or CAR T cells, genetically engineered T cells expressing specific CAR, which attach to tumor cells causing cell death. To evaluate CAR T cells' tumor-killing potential in vitro, begin with a brain tumor spheroid culture. Treat the culture with a suitable enzyme solution to obtain a single-cell suspension. Centrifuge and resuspend the cell pellet in a suitable media.
Pipette the desired volume of tumor cells followed by CAR T cells in a multi-well plate. In culture, specific receptors on CAR T cells recognize and bind to specific antigens on tumor cells. This binding activates CAR T cells and releases cytokines, resulting in tumor cell death. The activation also leads to increased CAR T cell proliferation.
Replace the media with an equal volume of tumor cells. Repeat cell addition on alternate days. Following repeated antigen exposure, few CAR T cells may undergo cell exhaustion. These cells display limited cytokine production and proliferation capabilities with increased inhibitory receptor expression. Harvest the cells from the plate and centrifuge.
Resuspend the cell pellet in FACS buffer containing an antibody cocktail. The antibodies bind to their target receptors on these cells. Add a fluorescent DNA-binding dye to stain the nuclei of permeabilized cells. Using flow cytometry, determine the different populations of cells via analysis of fluorescent cell surface markers.
Begin by dissociating glioblastoma tumor spheres from a glioblastoma tumor sphere culture with 1 milliliter of cold accutase and 30 to 60 seconds of pipetting. When the tumor spheres have been disrupted, stop the reaction with 5 milliliters of warm co-culture medium and pellet the tumor cell suspensions by centrifugation.
Resuspend the glioblastoma cells to a 1.6 times 10 to the fifth tumor cells per milliliter of fresh co-culture medium concentration and dilute T cells harvested from a CAR T cell culture to the appropriate percentage of CAR positive T cells per milliliter of co-culture medium concentration.
Next, add 100 microliters of the diluted tumor cells to each well of a 96-well flat-bottom tissue culture plate and 100 microliters of diluted CAR T cells into each well of tumor cells with gentle mixing. Then, place the plate in a 37 degrees Celsius, 5% carbon dioxide incubator.
On days 2, 4, and 6 of culture, carefully remove 50 microliters of medium from each well of the tumor cell T cell co-culture plate slated for rechallenge according to the table. Then, add 50 microliters of fresh glioblastoma cell suspension prepared as just demonstrated, but with twice the cell number of the initial co-culture to each well with gentle mixing, and return the plate to the cell culture incubator.
Days 1, 3, 5, and 7 of co-culture, transfer the supernatant from each well to be harvested into a new 96-well round-bottom plate according to the table and add 50 microliters of pre-warmed 0.5% trypsin-EDTA into each medium depleted well. After 5 minutes at 37 degrees Celsius, confirm that the cells have detached from the bottom of each well by light microscopy and gently pipette the enzyme solution around the bottom of each well to resuspend the cells before transferring the detached cell suspension into the appropriate corresponding wells of the new 96-well plate.
Pellet the cells by centrifugation and wash the cells with 200 microlitres of fluorescence-activated cell sorting staining solution, or FSS, per well with a second centrifugation. Resuspend the pellets in 100 microliters of the antibody cocktail of interest in 100 microliters of FSS per well for a 30-minute incubation at 4 degrees Celsius.
At the end of the incubation, remove any unbound antibody by sequential 100 and 200 microliter FSS washes and resuspend the cells in 100 to 200 microlitres of DAPI nuclear stain and FSS. Then, analyze the cells according to standard flow cytometric analysis protocols.
繁體中文
