The overall goal of this procedure is to generate cord blood derived cytotoxic T lymphocytes or CTL that recognize antigens from multiple viruses. This is accomplished by first isolating the cord blood mononuclear cells from the 20%fraction of the cord blood unit. The second step is to use the mononuclear cells to generate dendritic cells, Epstein-Barr virus, lymphoblasts cell lines, or E-B-V-L-C-L and the virus specific CTL.
The mononuclear cells, which contain the T cells, are stimulated with cells that have been transduced with the A five F 35 PP 65 vector. The final step is to confirm the specificity of the multi virus specific CTL and to freeze the CTL. Ultimately, the specificity of the expansion of the CTL for at least one viral antigen can be evaluated through Ellie SPA analysis and cytotoxicity assays.
Generally, individuals new to this method will struggle because the volume of core blood available in the 20%fraction is very small, which means that there are very few cells to work with. The procedure is also quite intricate and involves a generation of at least three different cell types from these limited cell numbers. We first had the idea for this method after we analyzed the data from a clinical trial where we made multi virus specific T cells for patients after bone marrow transplant.
In that study we saw that the T cells were not only safe but efficacious in these patients and prevented and treated viral infection. However, the patient population who have the greatest need for such a therapy are recipients of cord blood transplants and these patients, however, we are unable to go back to the donor and to get blood to make T cells. So we had to develop a novel strategy to generate multi virus specific T cells from the cord blood from the 20%fraction of the cord blood that would not be needed for the transplant procedure.
To begin warm 20 milliliters of RPMI in a 50 milliliter centrifuge tube and thaw the 20%fraction of cord blood. Then insert the spike of a female lure adapter into the outlet port of the 20%fraction and attach a syringe. Next, remove the blood and transfer it into the prepared centrifuge tube.
Rinse the cord blood bag with five milliliters of RPMI and then transfer the wash to the same tube. Now centrifuge the cells for 10 minutes at 400 Gs at room temperature and then after aspirating the re suspend the cells in 20 milliliters of warm RPMI transfer 15 milliliters of lymph prep to a 50 milliliter centrifuge tube. Layer the resuspended cells on top of it and separate the cells for 40 minutes at 400 Gs.Harvest the interface.
Then after washing the mononuclear cells in 20 milliliters of RPMI for 10 minutes at 450 GS and aspirating the supernatant, resuspend the pellet in 20 milliliters of RPMI count the cells and then after pelleting the cells again for five minutes at 400 Gs and aspirating the supernatant once more, resus suspend the cells at five times 10 to the six mononuclear cells per milliliter in serum free cellgenic dendritic cell media. Finally transfer one milliliter of the cells into a 15 milliliter conical tube for the E-B-V-L-C-L. Generation incubate two milliliters per well of a six well plate of the just prepared mononuclear cells for one to two hours after the incubation.
Wash off the non-adherent cells three to four times with PBS collecting the floating cells into a 50 milliliter centrifuge tube each time. Then freeze the collected cells at a controlled rate of one degree per minute and store in a minus 80 degrees Celsius freezer overnight. The next day transfer the cells to liquid nitrogen storage until the DC are ready.
Now add two milliliters per well of dendritic cell media supplemented with IL four and G-M-C-S-F to each well of the six well plate replenish the cytokines three to four days after DC initiation, adding the IL four and G-M-C-S-F in a total volume of 100 microliters of media to each. Well harvest the DC on day five to six by scraping the wells with a transfer pipette count the big cells and then after pelleting the collected cells for five minutes at 400 Gs and aspirating the supernatant resuspend the DC at two times 10 of the six cells per milliliter in DC media add half a milliliter of the cell solution per well into a 24 well plate. Then transduce the DC by adding the add five F 35 PP 65 vector at an MOI of 10 infectious units per cell to each of the wells of the 24 well plate.
Finally incubate the plate for 1.5 hours after the incubation at 1.5 milliliters of DC media supplemented with the DC maturation cytokine cocktail. Spin down the reserved mononuclear cells, then add 200 microliters of concentrated EBVB 95.8 supernatant and 1.8 milliliters of complete media supplemented with cyclosporine to the pellet. Next aliquot 100 microliters of the cells into 10 wells of a 96 well plate and 200 microliters five wells.
Add 100 microliters of complete media plus cyclosporine to the wells containing only 100 microliters of cells, and then fill the remaining wells with sterile water. Feed the LCL weekly. Eventually splitting the expanding cell cultures into T 75 flasks.
After harvesting the seven day old DC irradiate the cells at 30 gras. Then after washing them four times with PBS count, the DC resus suspend the DC in T-cell media containing human serum at one times 10 to the fifth DC per milliliter and then thaw an Eloqua of the previously frozen non-adherent cells after washing in T-cell medium and centrifuging for five minutes at 400 Gs.Count the thought cells and resus suspend them at two times 10 to the six cells per milliliter in T-cell media supplemented with human serum add IL seven IL 12 and IL 15 to the cells and then add one milliliter of cells to each well of a 24 well plate. Next, add one milliliter of the DC to each well containing non-adherent cells.
Fill any empty wells of the plate with sterile water and then incubate the T cells. After feeding the T cells on day seven, freeze the T cells nine to 12 days after T-cell initiation until the LCL are ready. Once the LCL have been passaged into the T 75 flasks, add the vector to the cells after they've been spun down at an MOI of 100 infectious units per cell and incubate the cells with the virus for 1.5 hours after the incubation.
Re suspend the cells in complete media at five times 10 to the fifth LCL per milliliter and add two milliliters of the transduced cells per well to a new 24 well plate after two days. Harvest the LCL and irradiate them at 40 gras after washing the cells four times with PBS and centrifuging at 400 Gs for five minutes. Count them and then resuspend the LCL in T-cell media containing human serum at 2.5 times 10 to the fifth cells per milliliter.
Now either add one milliliter of LCL per well of a new 24 well plate or add five times 10 of the sixth LCL to a GRE 40 culture device. Supplementing the culture with IL 15, harvest the T cells after centrifusion the T-cells for five minutes at 400 Gs and aspirating the supernatant reus. Suspend the cells in T-cell media supplemented with human serum at one times 10 of the six T-cells per milliliter.
After adding IL 15, either add one milliliter of the T-cell per well to the LCL in the 24 well plate or one milliliter of T-cell to the LCL in the GRE X 40 culture device. If a GRE X 40 is used, bring the total volume of media up to 30 milliliters. Finally on day seven, harvest and wash the CTL and LCL and irradiate the LCL as before.
Then after Resus suspending the LCL and T-cell media supplemented with human serum at 2.5 times 10 of the PIP cells per milliliter add one milliliter of LCL per well of a 24 well plate add T cells and supplement with IL two rather than IL 15 after three stimulations. Typically the majority of the cells are CD three positive with a mixture of CD four positive and CD eight positive T cells. There also are typically less than 15%of CD three negative CD 56 positive natural killer cells and less than 1%of CD 19 positive B cells.
The expanded CTL recognized the antigen PP 65 from CMV Heon and Penton from adenovirus, as well as numerous EBV antigens that are expressed on E-B-V-L-C-L. When tested in an Ellie SPA assay, CTL secrete more interferon gamma in response to these antigens than irrelevant antigens. As expected in a chromium release assay, CTL lys L-C-L-C-M-V-P-P 65, pulse and adenovirus, heon and or Penton pulse targets, but not targets pulse with irrelevant peptides.
While attempting this procedure, it's important to remember that all cells will be used in the manufacturing process and that cell numbers are extremely limited. It's also important to remember that because you are dealing with naive T cells, that you must always use human serum because the use of fetal calf serum might result in the generation of T-cell specific for fetal calf proteins. By following this procedure, tumor specific T cells or other virus to T cells likely can be generated as well.
