We optimized a highly qualified and clinically applicable method for the expansion of PBMC-derived cytokine-induced killer T cells and for evaluation of their cytotoxicity against cancers. The advantage of this technique is to provide a standard operating procedure for technicians and clinicians to quantify the potency of cytokine-induced killer cells in both scientific research and clinical evaluation. Begin this procedure with a preparation of CIK cells as described in the text protocol, then wash the CIK cells with 10 milliliters of sterile PBS.
Centrifuge for 10 minutes at 300 times G and 18 to 20 degrees Celsius. Aspirate the supernatant and resuspend the cells with five milliliters of PBS. Count the cell numbers and test cell viability using the trypan blue exclusion assay.
Aliquot the CIK cells into six steril 1.5 milliliter tubes at a density of approximately 5 to one million cells per milliliter of PBS. Label and treat the cells as detailed in the text protocol. Gently mix the CIK cells with the antibodies by gently pipetting up and down at least three times with a one milliliter steril pipette.
Incubate the cells for 15 minutes at room temperature in the dark. Centrifuge the tubes for 10 minute at 300 times G and 18 to 20 degrees Celsius. Following centrifugation, aspirate the supernatant and resuspend the cell pellet with one milliliter of PBS, then gently pipette the cells up and down at least three times with a one milliliter sterile pipette.
Transfer the cell suspension to a sterile five milliliter polystyrene round bottom tube with a cell strainer cap by gently pipetting through the cap, then place the tubes on the carousel in order. Open the flow cytometry analysis software and create an experimental folder, then click the new specimen button to add a specimen and tube to the experiment. Name the tubes as listed in the text protocol.
To create a scatter gating system for the CIK cell populations, first select tube one and click on the dot plot button to create an FSCA SSCA plot. Draw a rectangle gate over the entire cell population with an FSCA threshold greater than 5000 to exclude cell debris. Select the FSCA FSCH parameter for the new dot plot and draw a polygon gate around all single cells.
Select the count versus FITC-conjugated CD3 and count versus APC-conjugated CD56 parameter for the new histogram plot. Select the FITC-conjugated CD3 versus APC-conjugated CD56 parameter for the new dot plot and draw a four quadrant gate to define the four subpopulations. Record the data from 20, 000 single cells in each specimen.
Click the load sample button to analyze the blank control sample first. Identify the whole CIK cell population by using the CD56 and CD3 channel parameters. Open the files containing the statistical values of the individual specimen to analyze CIK cell populations and reprint them into analysis files.
To perform the cytotoxic assay, coculture CIK and human chronic myeloid leukemia K562 cells by adding one milliliter of K562 cells to each well in a six-well plate at a density of 5 million cells per milliliter, then add one milliliter of basal medium with or without CIK cells to the six-well plate as listed in the text protocol. Mix the cell suspensions by gently pipetting up and down at least three times. Place the plate in the incubator for 24 hours.
Now, coculture CIK and ovarian OC-3 cells by adding one milliliter of basal medium with or without CIK cells to the six-well plate as listed in the text protocol. Mix the cell suspensions by gently pipetting up and down at least three times. Put the plate in the incubator for 24 hours.
Following incubation, harvest the CIK K562 cell suspension directly into a 15 milliliter sterile tube. To harvest both the suspension and adherence cells from the CIK OC-3 groups, first transfer the cell suspension to a 15 milliliter sterile tube. Wash the well with one milliliter of steril PBS, collect the PBS, and add it to the tube, then add 5 milliliters of cell dissociation enzyme solution and incubate for five minutes at 37 degrees Celsius.
Add one milliliter of the solution from the same tube to the corresponding well, and gently mix the cells by pipetting up and down at least three times with a one milliliter sterile pipette. Collect all the cells in the same tube. Centrifuge at 300 times G for 10 minutes.
Aspirate the supernatant and resuspend the cells in one milliliter of sterile PBS. Pellet the cells at 300 times G for 10 minutes, then aspirate the supernatant and resuspend the cells in 100 microliters of sterile PBS. Add five microliters of 7-AAD dye to the cell suspension.
Gently mix the cells by pipetting up and down at least three times with a one milliliter sterile pipette. Incubate for 10 minutes and leave in the dark before analysis. To perform the cytolytic capability assay, mix the cell suspension and repeat the preparation for flow cytometry.
Click the new specimen button to add a specimen and tube to the experiment. Name the tubes as listed in the text protocol. To create a scatter gating system for the cytolytic assay, first select tube one and click on the dot plot button to create an FSCA SSCA plot.
Draw a rectangle gate over all of events with an FSCA threshold greater than 50, 000 to exclude cell debris. Select the SSCA CFSE parameter for the new dot plot, then select the 7-AAD CFSE parameter for the new dot plot and draw a four quadrant gate to define the four subpopulations. Click the load sample button to analyze the blank control sample first.
Adjust the voltage of SSCA and FSCA. Identify the dead cell population by using the CFSE and 7-AAD channel parameters. Record the data from greater than 20, 000 CFSE positive cells in each specimen.
Open the files containing the statistical values of each individual specimen to analyze the nonviable cell populations, and export the data into analysis files. The representative results of the gating strategy for analyzing the subpopulation of CD-3 positive CD56 positive T-cells from healthy donors are shown with the statistical analysis of the CIK proportion from three individuals. The CD-3 positive CD56 positive cell proportion significantly increased after 14 days of expansion.
This is evident through the 65%for the original PBMC which increased to 27.4%for the CIK cells harvested on day 14. In this culture system, the CIK cells yielded about half a hundred fold changes compared to the original number of PBMCs. K562 cells were stained with a nonfluorescent dye CFSE, which is cleaved by intracellular esterases within viable cells and becomes a highly fluorescent dye.
The size and granularity of the CIK and CFSE-positive cells are illustrated. The CFSE stained K562 cells were co-treated with CIK cells at a ratio of zero to one, five to one, and 10 to one. These cells were stained with 7-AAD dye as a viability probe for dead cell exclusion.
The 7-AAD positive cells of CFSE positive K562 cells were all evaluated. The CFSE-stained OC-3 cells were co-treated with CIK cells at a ratio of 10 to one. The obvious cytotoxicity of CIK against OC-3 cells is demonstrated here following 24 hours of incubation.
CIK T-cells has been reported to exert significant cytotoxic effects against cancer cells and reduce the adverse effects of surgery, radiation, and chemotherapy in cancer treatments. Immunotherapy is a promising treatment for a number of cancers. CIK can be efficiently expanded in vitro by incubation of patient-derived PMBCs in GMP-grade condition for further autologous infusion.
Following this protocol, we could execute the immune cellular therapy in a GTP-or GMP-grade facility for further clinical cancer treatment.
