This experimental system offers a high throughput methodology for monitoring T-cell mediated cytotoxicity at the single cell level. First, label the effector T cells and tumor target cells with fluorescent dyes dies. Load both cell types onto the nano well arrays and immerse the entire chip in medium containing cyt green and fluorescently labeled nexin.Five.
In order to identify both necrotic and apoptotic cells, now record an initial pre-image to determine well occupancy and cell viability at the start of the assay. After a six hour incubation record a second image. Then use automated image analysis to quantify the antigen specific lysis of EL four CD 19 positive by car positive T cells.
Results obtained from this high throughput method can screen antigen specific lysis of targets by effector cells based on positive staining with cytoxin.Five. The main advantage of this technique over existing methods like chromium 51 release assay is that true one-to-one effective target interactions can be achieved and the satellite frequency measured The implication of this technique extend to what therapy of cancer because it allows to study correlates of efficacy in autologous cell therapy To fabricate nano wells using the silicon master first thoroughly mix the cigar 180 4 elastomer kit base and curing agent at a 10 to one weight ratio in a disposable cup using a plastic knife. Degas the mixture in a vacuum chamber for one hour.
Then pour the mixture onto the silicon master and let it sit for 30 minutes. Now to cure the elastomer, heat the assembly in an 80 degree Celsius oven for two hours after cooling at room temperature for an hour. Carefully remove the PDMS nano well array off the silicon master.
Cover it with scotch tape until use a day before the experiment passage 5 million target cells in five milliliters. The day of the experiment. Pellet 5 million cells in a sterile 15 milliliter conical tube by centrifugation at 340 Gs for five minutes.
Aspirate excess media leaving behind approximately 50 microliters of media to label the cells. Add 150 microliters of the fresh cell tracker red working solution and mix thoroughly using a P 200 pipette incubate for 20 minutes at 37 degrees Celsius with 5%carbon dioxide. Using a hemo cytometer enumerate the effector cells harvested from culture.
Then pellet 5 million cells in a sterile 15 milliliter conical tube by centrifugation. Aspirate the excess media. Then add fresh working solution of five micromolar vibrant D cycle violet stain and resus suspend using a P 200 pipette.
Incubate the cells and die for 20 minutes at 37 degrees Celsius with 5%carbon dioxide at 6.8 milliliters and 6.0 milliliters of R-P-M-I-P-L-G-H to target and effector cells respectively and mix by pipetting. Then slowly layer 3.5 milliliters of fial pack plus to the bottom of each tube, centrifuge both tubes at 340 GS for 30 minutes with no break and acceleration during centrifugation, prepare the nano well array First transfer seven milliliters of pre-war PBS to a four well plate. Clean the bottom of the glass slide with ethanol using a plasma oxidizer.
Treat the PDMS at high RF setting for 30 to 45 seconds. Then place the array into the PBS. Transfer the plate to a biosafety cabinet and aspirate excess PBS apply warm 1%noble agar on the top and bottom edges of the glass slide to immobilize the array.
Ensure that you do not let the PDMS dry after 10 minutes at three milliliters of PBS and aspirate the excess agar. Then add three milliliters of R 10 onto the top of the nano well array and equilibrate for about 10 minutes. Subsequent to Fial centrifugation aspirate five milliliters of media from the top layer of each tube.
Being careful not to aspirate the layer containing the cells with a P 1000 pipette. Harvest the white layer of cells between the fial and the media. Transfer the cells to new sterile conical tubes.
Now wash the harvested effector and target cells twice with three milliliters of pre-war R-P-M-I-P-L-G-H. After the final centrifugation aspirate the excess media and resuspend the cell pellet. In 500 microliters of R 10, count the viable cells using the trian blue exclusion method on a hemo cytometer aspirate the excess media from the nano will array.
Then add two milliliters of fresh R 10 aspirate. Again, making sure the top of the nano will array is neither too wet nor too dry as it will affect distribution of the cells. Now deposit one times 10 to the fifth of fector cells in 200 microliters of R 10 onto the nano will array surface allowed the cells to settle for five minutes.
Then using a standard tissue culture microscope, verify the desired distribution of the cells. Next deposit one times 10 to the fifth target cells in 200 microliters of R 10 onto the nano well array surface containing the effector cells. After five minutes, evaluate cell distribution by microscopy.
Then rinse the nano well array carefully with two milliliters of R 10 and aspirate excess media in a separate sterile 15 milliliter conical tube. Pipette three milliliters of prewarm R 10 at three microliters of 0.5 millimolar cyt, green nucleic acid stain, and 60 microliters of an X in five LOR 6 47 mixed thoroughly by pipetting. Now gently pipette the stain solution onto the four well plate, making sure that the cells are not displaced from the wells incubate for 15 minutes at 37 degrees Celsius, 5%carbon dioxide acquire images of the nano well array using a fluorescence microscope like the zes observer Z one microscope, equipped with a motorized stage and Lambda DG four.
Initialize the software and the microscope. Check the signal intensity on the transmitted light and the other fluorescent channels. Adjust the microscope setup to ensure maximal signal to noise while avoiding saturation.
Now open the x and Y position list for the nelle array. Set the XY zero position and zero the focus on the center of a seven by seven nelle array Block on the top left corner of the nelle array stamp. Set the XY positions to coincide with the center of each block and the Z value to accurately reflect the focus on each block.
Now set the image acquisition in linear mode and acquire images. After the first imaging series, return the nano well arrays to the cell culture incubator for six hours. Taking care of the agitation can lead to cell displacement.
Then repeat acquisition of the images at the second time point. This high throughput cytolytic assay measures the frequency of antigen specific lysis labeled CD 19 specific CAR positive T-cells were co incubated with labeled mouse EL four target cells in the individual wells of a nano L array. The target cell histogram plots represent wells containing a single effector cell and a single target cell.
After six hours of co incubation matched histograms of target cells without effectors were used as negative controls to report the frequency of background cell death. It is desirable to achieve low frequencies of non-specific lysis in the range of two to 4%The nano well array can be adapted for time-lapse imaging to allow for the observation of effector, target conjugation, and subsequent cell death. Here unlabeled car positive T cells were co incubated with NA LM six GFP target cells in an NOL and subjected to dynamic monitoring.
When an Xin five was added to the culture media, the apoptotic cells label red Once mastered. This technique can be performed properly in 10 to 12 hours Following this procedure. Other methods like micro grabbing can be used to determine the cytokine secretion profile of the cells.
