GMP protocol to gene-engineer and process human T-cells revisited: medium critically determines yield, function and differentiation state of CD8+ T-cells

We have previously designed and validated a GMP protocol for retroviral gene transduction and expansion of human T-cells. Here, we have revisited the choice for optimal medium (composition) and tested different and newly available media for their effects on yield as well as phenotypical and functional properties of receptor-engineered T-cells. GMP-defined (serum-free) media that were tested included AIMV and Optimize (Invitrogen), Xvivo15 (Lonza), CellGro (CellGenix) and TexMACS(Miltenyi). We tested these media with or without supplementation of 2% plasma. In addition, we tested 3 blended media (i) AIMV (20%)+RPMI(80%)+2% plasma (EMC standard); (ii) AIMV(50%)+RPMI(50%)+5% human serum; and (iii) Xvivo15(20%)+RPMI(80%)+2% plasma. First experiments used CAR-engineered T-cells.The non-supplemented media performed the least with respect to T-cell yield, transduction efficiency and function of CAR T-cells. The best performing media with respect to these parameters included 3 plasma-supplemented media (AIMV, Xvivo15 and TexMACS) and the 3 blended media, including the EMC standard. Five of these media, supplemented with IL15+IL21, were re-tested in parallel to generate MC2/A2 TCR T-cells. In terms of T-cell yield, transduction and function, AIMV+2% plasma performed the best, whereas Xvivo+2% plasma performed the least. Notably, with regard to the preservation of early T-cell differentiation this sequence was reverse. Taken together, this study shows the impact of culture media on yield and phenotypical and functional properties of human T-cells. Importantly, the medium resulting in highest T-cell yield led to more differentiated T-cells, and vice versa. This study provides important information with respect to medium selection to gene-engineer and process T-cells for clinical studies.