De-lib-er-ating a CAR T Cell product with functional competence that engraft, proliferate and has cit-el-uric activity following infusion is a key component of an effective adoptive immunotherapy. For example, in solid tumors, CAR T cells they can penetrate solid tumors and they receive optimal antigen stimulation;however, their overall engraftment and proliferation is impeded in people. The main benefit of this approach is that it can retain the quality of the CAR T cells or differentiation state of the CAR T cells without generating terminally differentiated or exhausted T cells, which would have diminished proliferative capacity or poor effective function following infusion.
This technique can be difficult because T cells are very sensitive to their cell concentration or the surface area of the vessels that their cultured in. As well as their metabolic needs. Essentially T cells need to be kept happy throughout the process, otherwise they won't grow.
To begin this procedure, set out 6-well culture dishes. Activate fresh or cryo-preserved primary human T cells by mixing them with anti-CD3 CD28 magnetic beads, at a ratio of three beads per T cell in the wells of the culture dishes. Culture the T cells in X-Vivo 15 medium supplemented with normal human AB serum, L-glutamine, he-pees and IL2.
Activate the T cells at a concentration of one million T cells per milliliter during expansion. And culture them at 37 degrees Celsius with 20%oxygen, 5%carbon-dioxide and 95%humidity. After overnight stimulation, add lentiviral supernatants to the activated T cells.
Calculate the volume of supernatants necessary to achieve a multiplicity of infection between three and five. Continue culturing the cells using the same conditions. On day three, collect a representative aliquot of the cells of cryo-preservation.
Prior to cryo-preservation, remove the magnetic beads by gently pipetting and magnetic separation. Prepare the freezing medium, which contains PBS with 0.5%DMSO. And store it at four degrees Celsius until ready to use.
Next, centrifuge the T cells at 300 times G for five minutes. Discard the supernatant and add five milliliters of PBS. Centrifuge the cells again at 300 times G for five minutes and discard the PBS.
We suspend the pellant in one milliliter of cold cryo-preservation medium. Freeze the T cells in a chilled freezing container. And store at minus 80 degrees Celsius for 48 hours.
After this, transfer the frozen cells to liquid nitrogen. Wash the rest of the T cells once in five milliliters of PBS to eliminate any residual vector. Centrifuge at 300 times G for five minutes.
Then, decant the PBS and re-suspend the cell pellant in T cell culture medium at a concentration of 500, 000 cells per milliliter. Split the T cells into two cultures designed 45 and nine. Count the T cells by flow cytometry using counting beads and monoclonal antibodies to human CD4 and CD8.
As well as a viability dye. Refeed the cultures every other day to maintain them at a concentration of 500, 000 cells per milliliter. On day five, count and cryo-preserve the day five cultures as previously described.
On day seven, wash 500, 000 cells in PBS. And re-suspend them in 100 microliters of fluorescence activated cell sorting buffer. Then, detect CAR surface protein expression by immuno-staining with a fluorescently conjugated anti-CAR 19 idio-type by flow cytometry.
On day nine count and cryo-preserve the day nine cultures as previously described. In this study the function and efficacy of CAR T cells that are harvested at varying intervals throughout X-Vivo culture are measured. Using the methods described in this video, T cells are stimulated and expanded for either three or nine days.
The cells differentiation profile as indicated by the gating strategy shown here, is analyzed by measuring the abundance of distinct glycoproteins expressed on the cell surface. A progressive shift towards effector differentiation is seen over time during X-Vivo culture. The effector function and proliferative capacity of CAR T cells in response to antigen is then assessed.
Cells that are expanded less are functionally superior to those extensively cultured over a longer duration. In a human xenograft mouse model of ALL the potency of CAR T cells harvested at different time periods is compared. The nine day cells show a dose-dependent anti-lukemic response with a complete response worth a high dose of three million;and a loss of efficacy for the low dose of 500, 000.
However, the day three cells showed persistent tumor control in both high and low doses. This response is associated with the absolute count of CAR-T 19 in the peripheral blood of mice. Overall, these results provide evidence that CAR T cells harvested earlier, outperform those harvested later.
Following this procedure, additional function assays such as seahorse assay can be performed to measure their metabolic properties such as oxygen consumption or spir-it-ed capacity. Several rounds of re-stimulation can also be performed to provide insight into their differentiation and persistence of CAR T cells. Using this work as a foundation we and other researchers are engaged in studies to abbreviate the culture process further.
It can also be extended in core approaches design for other cancer antigens. Care always have to be taken when you work with lentivirus. But even when the lentivirus is engineered to be replication incompetent.
