Fig. 3

Optimized process for enhanced expansion and functionality of mesoCAR-T from cryopreserved PBMCs. (A) Optimization of electroporation density. (B) Optimization of culture density after electroporation. Viability (C) and expansion potential (D) during mesoCAR-T manufacturing. (E) Percentages of CD3⁺, CD4⁺, CD8⁺, and mesoCAR⁺ cells (n = 3). (F) Differentiation and exhaustion states (n = 3). (G) Proportions of apoptotic cells and CD3⁺, CD4⁺, CD8⁺ subsets expressing CD45RO^-CD27⁺ (n = 3). (H) The secretion level of PD-1 at an E:T = 2:1 (n = 3-4). (I) Cytokine secretion (n = 3). (J) The cytotoxic capacity curves at different E:T ratios (4:1 or 2:1) against SKOV-3 cells, and the cytotoxicity at 4 h and 24 h is shown in the histogram. Data is presented as median with 95% CI. Each dot represents an individual. Statistical analysis was conducted using the linear regression (A), Kruskal–Wallis test (C, D, J) or Mann Whitney test (B, E, F, G, H, I), with a p value < 0.05 indicating significance. * p < 0.05, “ns” indicates no significant difference.