Quantification of CAR-T cells by flow cytometry demonstrated that transduction with both LV supernatants produced in serum-free medium (L-TEX and PJ-TEX) more than doubled the frequency of CAR+ cells compared to the L-DMEM condition (L-DMEM = 30.77%☐.04%, L-TEX = 76.01%☐.25%, PJ-TEX = 73.54%☑.05% CAR+cells). LV produced by Lipofectamine transfection and harvested in DMEM 10% FBS (L-DMEM group) were used as controls. To that end, we generated CAR-T cells by transducing human T-cells with LV particles harvested in TexMACs medium after transfection with Lipofectamine (L-TEX group) or PEI JetOptimus (PJ-TEX group). Finally, we evaluated if these tested variables in producing LV particles would affect transduction efficiency. Viral titer resulting of lipofectamine-based transfection in the same conditions was equivalent (1.29×10 7 ± 1.30×10 6 IU/mL). The yields were further increased when the DNA amount was reduced to 104 ng/cm 2 and the producer cell line was changed to LentiX-293T (1.32×10 7 ± 0.13×10 7 TU/mL). We found that among the PEI formulations, transfection with PEI JetOptimus resulted in the highest LV titers. In addition, viral titers were decreased as the amount of PEI was increased. ![]() The infectious titers obtained from calcium phosphate transfection method were the lowest among the tested methods. The resulting LV titers were compared to that obtained with lipofectamine-based transfection. Next, we compared the three most used DNA-delivery methods: calcium phosphate precipitation, cationic lipids (Lifofectamine™) and polycations (linear PEI 25 KDa, PEIPro and PEI JetOptimus). Among the media tested, harvesting in TexMACS and DMEM 10% FBS yielded the highest viral titers (4×10 6 transducing units per mL or TU/mL). For this purpose, HEK293/T17 cells were transfected with lipofectamine and viral titers were measured by flow cytometry. In addition, we included DMEM 10% FBS and FreeStyle media which are commonly used for viral production for comparison. Our hypothesis was that collecting LV particles in a medium optimized for T-cell growth, such as TexMACS and X-VIVO 10, would improve transduction efficiencies. Initially, we evaluated the LV titers harvesting in different media: TexMACS™, X-VIVO™10, FreeStyle™ and DMEM supplemented with 10% fetal bovine serum (FBS). Here, we demonstrated that an improvement in lentiviral production can be achieved by optimizing parameters such as serum-free media for lentivirus (LV) harvesting, PEI formulations, DNA amount, PEI:DNA ratios and producer cell lines. ![]() Optimizing the production of vectors for CAR-T cell transduction before beginning large-scale manufacturing reduces variability and maximizes efficiency. ![]() Production of CAR-T cells requires several carefully performed steps and consistent production of optimal viral vectors is a critical step to ensure efficient gene modification of lymphocytes. Chimeric antigen receptor (CAR) T cell therapy is a new approach for treatment of cancer.
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