Static culture in 96-well U-bottom

Protocol: CD3⁺ T cells were purified from healthy human donors (n=8; EasySep™ Human T Cell Isolation Kit, Stemcell Technologies)*. Cells were activated with Dynabead Human T-Activator CD3/CD28 (Thermo Fischer) at a ratio of 1:1 bead:cell in CellGenix® GMP TCM or competitor media in the presence of IL-7 and IL-15 (10 ng/ml, Sartorius CellGenix). Media and IL-7 and IL-15 (10 ng/ml, Sartorius CellGenix) were added on day 4 and day 7. Cells were splitted on day 4 and day 7 and harvested on day 9.

*In some experiments, cells were frozen in CellGenix® GMP TCM with 10% DMS after purification and thawed before activation.

Figure 1: T cell expansion with CellGenix® GMP TCM (day 9)

CD3⁺ T cells purified from human donors (n=8) were activated using anti-CD3/anti-CD28-coupled magnetic beads in CellGenix® GMP TCM or commercially available media for T cell expansion in the presence of IL-7 and IL-15 (10 ng/ml, Sartorius CellGenix).

Figure 2: T cell viability on day 9 of culture
CD3⁺ T cells were cultivated as described. On day 9 cells were labeled with 7-AAD and viability was determined by flow cytometry. Median and spread of n=8 donors are displayed.

Figure 3: Proportions of CD4⁺ and CD8⁺ T cells
CD3⁺ T cells were cultivated as described. On day 9 expression of CD4 and CD8 was determined by flow cytometry (n = 8 donors).

Figure 4: Memory phenotype after 9 days expansion in CellGenix® GMP TCM or competitor media
CD3⁺ T cells were cultivated as described. On day 9 the expression of memory cell markers was determined by flow cytometry. For each memory T cell subpopulation, the median of n = 8 donors is depicted. T_CM, central memory T cell; T_SCM, memory stem T cell; T_EM, effector memory T cells; T_EMRA, terminally differentiated effector T cells.