Establishment of a serum-free culture system with an optimized 5-interleukin cytokine cocktail and insulin to promote preferential CD8⁺ T-cell expansion

Adoptive T-cell therapy (ACT) requires the robust ex vivo expansion of functional T cells, but serum-containing culture systems, although supportive of proliferation, remain unsuitable for standardized clinical manufacturing. This study established a defined, serum-free culture platform that promoted effective CD8⁺ T-cell proliferation. CD3⁺ T cells were purified from mononuclear cells and cultured under various cytokine and medium conditions. Using fractional factorial screening followed by steepest ascent optimization, we identified an optimal 5-cytokine cocktail (Step 7 formulation: 30.8 ng/mL interleukin [IL]-2, 67.3 ng/mL IL-4, 40.3 ng/mL IL-7, 41.2 ng/mL IL-10, and 70 ng/mL IL-15), which markedly enhanced T-cell proliferation and increased the proportion of CD8⁺ T cells. Evaluation of basal media under serum-free conditions revealed that X-VIVO 15 effectively supported T-cell expansion in the presence of the cytokine cocktail. When insulin (4.39 µg/mL) was added to X-VIVO 15 together with the Step 7 cocktail, CD8⁺ T-cell proliferation reached levels comparable to those achieved in 10% fetal bovine serum cultures while maintaining serum-free conditions. This optimized system provides a reproducible and scalable platform for serum-free expansion of human T cells, supporting the manufacturing of ACT and chimeric antigen receptor T-cell therapies.