xCELLigence real time cell analysis enables you to quantitatively monitor the killing of cancer cells by immune cells under label-free conditions and over the course of multiple days. This system provides a complete time course of target cancer cell killing allowing the simultaneous screening of a large number of constructs, effector cell types, or combination therapies at low effector to target ratios. Begin by seeding 1.5 times 10 to the seven HEK293FT cells in DMEM culture medium in a 150 milliliter cell culture dish for an overnight incubation at 37 degrees Celsius and 5%carbon dioxide with humidity.
The next morning, mix 2.5 milliliters of transfection dilution solution supplemented with five micrograms of lentiviral vector plasmid DNA and 22.5 micrograms of lentiviral packaging mix with 82.5 microliters of transfection reagent in 2.5 milliliters of transfection dilution solution. After a 15 minute incubation at room temperature, transfer the reaction drop wise into the dish of HEK293FT cells and return the cells to the incubator for a second overnight incubation. The next morning, replace the supernatant with 19 milliliters of fresh DMEM culture medium and return the cells to the incubator overnight.
Collect the supernatant into a single 50 milliliter conical tube for four degrees Celsius storage for the next two days before sedimenting the lentivirus by centrifugation on the second morning in order to remove any cells or debris from the solution. Transfer all but about one milliliter of the lentivirus containing supernatant to an ultra clear centrifuge tube for ultracentrifugation. After ultracentrifugation, carefully aspirate the residual virus containing supernatant, gently add 100 microliters of DMEM to the pellet, and place the tube on ice for 15 minutes.
At the end of the incubation, mix the solution gently and aliquot the lentivirus solution into pre-chilled sterile tubes for storage at minus 80 degrees Celsius. Then determine the titer of the lentivirus by quantitative RT-PCR according to the manufacturer's instructions. For CAR-T cell generation and expansion, activate one to two times 10 to the six peripheral blood mononuclear cells in one milliliter of CAR-T cell medium with an equal number of CD3/CD28 coated microbeads in one well of a 24-well plate in the cell culture incubator for 24 hours.
The following two mornings, mix one microliter of transduction enhancer agent with the cells followed by the addition of lentivirus at a multiplicity of infection of five to one. Monitor the T cell growth every two to three days adding fresh CAR-T cell medium as necessary to maintain the cells at a one to two times 10 to the six cells per milliliter concentration. To detect T cell CAR expression by flow cytometry, transfer three times 10 to the fifth CAR and non-transduced T cells into individual 1.5 milliliter microcentrifuge tubes.
Collect the cells by centrifugation and resuspend the pellets in 200 microliters of FACS buffer supplemented with 1%human serum. Split each cell sample into 100 microliter aliquots in individual five milliliter polystyrene FACS tubes on ice. After five minutes, add one microliter of biotinylated FAB2 fragments of goat anti-mouse FAB2 to one tube of each cell type and two microliters of PE-labeled anti-tag antibody to the other tube of each cell type with thorough mixing.
After 30 minutes on ice, wash the cells with three milliliters of fresh FACS buffer and after centrifuging decant the supernatants. Resuspend the pellets in the remaining supernatant and add two microliters of APC anti-CD3 and two microliters of 7-AAD to each tube. Add one microliter of PE-labeled Streptavidin with brief mixing to the tube stained with anti-FAB2 antibody and incubate all of the tubes on ice for 30 minutes.
At the end of the incubation, analyze the cells by flow cytometry according to standard protocols gating the T cells by forward scatter versus side scatter and CD3 versus 7-AAD expression. For a real-time cytolysis potency assay, add 50 to 100 microliters of target cell culture medium to each well of an e-plate and measure the background impedance in a cell analyzer instrument. Detect the target cancer cells from the culture plate using trypsin.
Wash the cells in 15 milliliters of fresh culture medium and centrifuge. Resuspend the cancer cell pellet in five milliliters of fresh medium for counting and adjust the cell density to the appropriate target cell concentration. Add the cells to the appropriate number of wells and leave the e-plate for 30 minutes at room temperature to allow the cells to settle evenly on the bottom of the wells.
At the end of the equilibration period, place the plate in the real-time cell analyzer and initiate measurement of the impedance automatically every 15 minutes overnight. The adhesion and proliferation of the target cells can be tracked in real time. The next morning, dilute the effector CAR and control T cells to the appropriate concentration in individual tubes and remove 50 to 100 microliters from each well of the e-plate so that the final volume in each well is 100 microliters.
Next, seed the effector and control T cells in the appropriate wells at the appropriate effector to target ratios in 100 microliters of medium per well. Then equilibrate the e-plate for 30 minutes at room temperature before returning the plate to the cell analyzer system. To measure the efficacy of the T cell mediated killing of the target cancer cells, monitor the CAR-T mediated killing for 96 hours.
About 50%of the CAR-T cells stained positive for anti-single chain variable fragment antibody indicating the expression of CAR in about 50%of the T cells. In this representative experiment, a 10 to one effector to target ratio was used for all of the groups. Raji cells only and CD22-CAR T cell treated Raji cells displayed significant killing compared to the control T cell only and Mock CAR-T cell groups.
In this experiment, only the CD47-CAR-T cells were effective at killing the pancreatic cancer cells. Further, the impedance signal from the CD47-CAR-T cells alone was only slightly above the cell index signal measured in the medium alone wells indicating that the impedance signal resulting from suspension cells such as CAR-T cells is minimal and does not contribute to the overall impedance signal when CAR-T cells are added to the target cancer cells. Notably, the GITR co-stimulatory domain was observed to be more effective in enhancing CAR-T cell killing against epithelial growth factor receptor positive target cells compared to original CAR constructs without the GITR domain.
The medium from e-plate wells can be collected to analyze the cytokine profile at time points either during or after the assay according to the needs of the experiment. Overall, xCELLigence has improved the efficiency of evaluating the potency of diverse immunotherapies ranging from bi-specific T cell engagers and oncolytic viruses to immune check point inhibitors in combination therapies.
