Begin by turning on the instrument and selecting the T-cell transduction process from the touchscreen interface. Click run to initiate the procedure and follow the onscreen instructions provided. On the perimeter input screen input the operator initials tubing set, lot number, and expiration date.
Next, select full process on the process setup screen. Now, choose two vials of selection reagent for the CD4, CD8 selection method. Install the tubing set, ensuring that all lure connections are tightly set and complete the upper and lower integrity tests by following the instructions on the touchscreen.
Now, attach the medium and processing buffer bags per the onscreen guidance and begin the automatic priming of the tubing set. Once the transfer cell product screen appears, transfer the thawed T-cell product to a 150 milliliter transfer bag and sterile weld it to the application bag of the tubing set. Next, use a quality control pouch to obtain a sample from the application bag and perform a cell count using a hematology analyzer.
Connect the CD4 and CD8 reagent vials to the tubing set and start the selection process for T-cell enrichment. After enrichment, remove a sample of the selected cells from the reapplication bags quality control pouch. Enter the desired cell concentration and starting number.
Now, attach a vial of the activation reagent according to onscreen instructions. Then, set the carbon dioxide concentration to 5%and the culture chamber temperature to 39 degrees Celsius. Establish the activity matrix using the enhanced feeding protocol as a starting point and modify individual steps to your specific optimized protocol.
In this case, set the transduction activity time to 24 hours after seeding and the culture wash to 48 hours after transduction. Set the activate shaker time to begin 30 minutes after starting the culture wash. Delete any medium bag exchange and waste bag exchange activities as they are no longer required.
Then, press okay on the screen to initiate cultivation. Allow the instrument to automatically pump the required volume from the reapplication bag into the culture chamber. Thaw and prepare the calculated vector volume in a 20 milliliter reagent bag with a cold medium to reach a final volume of 10 milliliters.
Adjust the activity matrix to set the transduction time to two minutes into the future and press okay to initiate the transduction activity. Sterile weld the vector bag to the tubing set following the onscreen instructions. Modify the activity matrix according to the actual transduction start time with the culture wash scheduled 48 hours after transduction.
Schedule the time of activate shaker to 30 minutes after the culture wash. Finally, ensure the media exchange occurs in the afternoon at 2:00 PM on day six. Press the sample button and follow the onscreen instructions to acquire a quality control sample from the active culture.
Divide the sample into three one milliliter aliquots for various analyses. Prepare the final formulation buffer, saving a portion for the cryoprotectant preparation later. Next, adjust the activity matrix, setting the end of culture to two minutes in the future.
Attach the final formulation buffer to the tubing set and begin harvest. Seal off and remove the target cell bag. This bag contains the CAR T-cells ready for infusion or cryo-preservation.
RT runs of the clinical trial was 46%This indicates that the TCT process effectively promoted cell expansion. The cell viability of the final product was between 88 to 94%and the CD3 T-cell purity was 89 to 93%Endotoxin, mycoplasma, sterility, replication competent lentivirus, and residual leukemic cells were not detectable.
