The overall goal of this procedure is to demonstrate the preparation of clinical grade dendritic cells for immunotherapy. This is accomplished by first freezing peripheral blood mononuclear cells isolated from leukapheresis. In the second step, the frozen aliquots of pbmc are thawed and the monocytes are differentiated into dendritic cells in the presence of G-M-C-S-F and IL four for five days.
Next, the dendritic cells are harvested, pulsed with peptides, and matured overnight with the TLR three agonist poly ICLC. During the final step, the mature dendritic cells are harvested and frozen in aliquots. These frozen aliquots of mature dendritic cells can be thawed, transferred into syringes and injected back into the patients as part of their immunotherapy.
Demonstrating the procedure will be merited sedia and farra Hassan, both research techs in my lab Using a plasma transfer set begin by aseptically spiking one of the access ports in the leukapheresis bag. Then use a 60 milliliter syringe to transfer the leukapheresis into a sterile 500 milliliter bottle. Then using room temperature RPMI adjust the volume of the leukapheresis to two times its original volume and mix thoroughly after gently mixing a bottle of fial pack plus add 12 milliliters of the solution into sterile 50 milliliter conical tubes, and then carefully layer 30 milliliters of the diluted leukapheresis product into each tube of fial.
Be careful not to disturb the interface between the FI call and the cell suspension. Centrifuge the tubes for 20 minutes at 1000 GS and room temperature with no break. Then carefully harvest the cloudy layer of pbmc from each tube and transfer it to new sterile 50 milliliter tubes.
Next, add RPMI to each tube to a final volume of 50 milliliters, and then mix the cell suspensions gently by inversion. After spinning down the cells for 10 minutes at 500 Gs and four degrees Celsius, remove the supernatants from each tube and resuspend the cell pellets from each tube in RPMI to a final volume of 50 milliliters. After mixing the tubes gently by inversion, determine the number and viability of the cells.
Next, centrifuge the cells and resuspend the pellet in freshly prepared freezing media at a final concentration of two times 10 to the eight cells per milliliter. Transfer one milliliter Eloqua of the cell solution into 1.8 milliliter cryo vials. Then transfer the cryo vials into a controlled rate freezer and begin the freezing run At the end of the run, transfer the frozen cryo vials immediately into the vapor phase of a liquid nitrogen freezer.
The next day haw the aliquots of frozen p BMCs in a 37 degree Celsius water bath by gentle agitation. When completely thawed, transfer the contents of the vials to sterile 50 milliliter conical tubes containing 30 milliliters of RPMI and autologous plasma centrifuge. The cells carefully resus suspend the cell pellet in a small volume of RPMI plus autologous plasma.
Then add more RPMI plus autologous plasma to a final volume of 50 milliliters. After mixing the suspensions thoroughly plate 1.4 times 10 to the eight cells in 40 milliliters of RPMI plus autologous plasma onto a 225 centimeter squared Easy flask incubate the easy flask flat on its side for one to two hours at 37 degrees Celsius in the tissue culture incubator containing 5%carbon dioxide to allow the monocytes to adhere to the flask when the incubation is complete. Wash the easy flask twice with 30 milliliters of pre-war RPMI to remove the non-adherent cells.
After the second wash, add 40 milliliters of RPMI plus autologous plasma with IL four and G-M-C-S-F to the cells, and then incubate the EASI flask for two days at 37 degrees Celsius and 5%carbon dioxide. Five days later vigorously swirl the flasks and pipet up and down to resuspend. The non-adherent and loosely adherent cells transfer the harvested cells to new 50 milliliter conical tubes.
After spinning down the cells, we suspend the cell pellets in 50 milliliters of RPMI plus autologous plasma. After mixing and washing the cell, suspensions again plate one to two times 10 of the six cells per well in three milliliters of RPMI plus autologous plasma with IL four and G-M-C-S-F in six. Well tissue culture plates then incubate the plates at 37 degrees Celsius in the tissue culture incubator containing 5%carbon dioxide overnight the next day at 10 micrograms per milliliter of KLH to one third of the wells in the six well culture plates and mix the plates by swirling.
Then add two micrograms per milliliter of poly ICLC to the wells and mix well. Next, add 100 micrograms per milliliter of long peptide antigens to their designated wells, each well receiving only a single peptide to avoid cross competition. Then incubate the plates overnight at 37 degrees Celsius in a tissue culture incubator containing 5%carbon dioxide on day seven, harvest and pool all the wells containing dendritic cells.
Pulsed with the peptides on day six into new 50 milliliter conical tubes. After spinning down the cells, resus, suspend the aspirated cell pellets in five milliliters of RPMI plus autologous plasma, and then bring the total volume up to 50 milliliters with RPMI plus autologous plasma and mix the cell suspensions thoroughly. After calculating the total number of viable cells, centrifuge the tubes resus suspend each pellet in five milliliters of sterile sodium chloride and then wash each cell suspension two more times in 14 milliliters of sterile sodium chloride.
After each wash, mix the tubes after the second wash. Resus suspend the cell pellets in dendritic cell freezing solution at four to 20 times 10 of the six cells per milliliter and aliquot one milliliter of each cell suspension into 1.8 milliliter cryo tube vials. Finally, using the controlled rate freezer, freeze down the aliquots of dendritic cell vaccines and then transfer the aliquots immediately into the vapor phase of a liquid nitrogen freezer for long-term storage.
Between 10 to 20%of starting p BMCs differentiate into dendritic cells at the end of the culture period. Maturation of the dendritic cells in response to poly ICLC represented by the bold lines was evaluated based on the expression of CD 83, CD 40, and CCR seven, and compared to unstimulated dendritic cells represented by the gray shades as demonstrated in these bar graphs, poly IIC matured dcs in this experiment differentiated from three healthy donors secrete large amounts of IL six IL eight IL 12, and TNF alpha. These poly ICLC matured cells also induce allogeneic T-cell proliferation as evidenced by CFSE expression, as well as interferon gamma production After its development.
This technique paved a way for researchers in the field of immunology to explore the use of dendritic cells for immunotherapy in cancer and infectious diseases.
