Fig. 4

Significant differences were observed in CAR-T manufacture using different T cell selection buffers. This figure compares DPBS or X-VIVO15 basic medium as the CAR-T manufacturing process’s CD3 + T cell selection buffer. The DoE was repeated three times, and three PBMC samples were used in each repeat. Groups and factor levels in the DoE are shown in Table 1. a and b show the percentage of CD3⁺ cells that express CD25 (a) or CD69 (b) surface markers. In c, the cell diameter was measured before CD3⁺ T cell selection and after 48-h T-cell activation. In a, b, and c, an unpaired t-test was used, and a two-tailed P value was calculated; ns, P > 0.9999; ****, P < 0.0001. d Changes in CD25 expression percentage in CD3⁺ cells, e CD69 expression percentage in CD3⁺ cells, and f live-cell diameter in the CAR-T manufacturing process. g Expansion fold of CAR-T cells after retroviral transduction. h CAR expression percentage in CD3⁺ cells at days six and nine after retroviral transduction