4cell nutri t medium

PBMCs were activated in plates coated with 1 µg/mL anti-CD3 mAb and cultured in the presence of 3 µg/mL anti-CD28 mAb, 25 U/mL human IL-7 and 50 U/mL human IL-15 for 2 days. Next, 8 × 10⁴ activated human PBMCs were incubated with 0.5 µL VSV-LV (MOI 4–5) in a flat-bottom 96-well plate or respectively upscaled for bigger production in a 24-well plate. Thereafter cells were cultivated for 24 h until short-term CAR T cells were harvested, washed and used for subsequent experiments. To evaluate the transition of short-term CAR T cells to fully CAR-expressing T cells, stCAR T cells were cultured in 4Cell® Nutri-T medium (Sartorius) supplemented with 25 U/mL human IL-7, and 50 U/mL human IL-15 for 3 additional days. GFP-transduced cells were produced exactly under the same conditions by replacing the packaged CAR by the GFP sequence.

Human PBMCs were isolated by Histopaque gradient centrifugation from freshly sampled human blood, purchased from the German blood donation center (DRK-Blutspendedienst, Frankfurt am Main). Blood donations were from anonymous donors. No ethics vote was required in this case as confirmed by the Ethics Committee of Goethe-University Frankfurt. Primary cells were cultured in 4Cell® Nutri-T medium (Sartorius) supplemented with 0.4% penicillin/streptomycin, 25 U/mL human IL-7 (Miltenyi Biotec) and 50 U/mL human IL-15 (Miltenyi Biotec). Monocytes were isolated from PBMCs by anti-human CD14 (REA599, APC, Miltenyi Biotec) antibody labeling and subsequent isolation using a magnetic anti-APC-Microbead Kit (Miltenyi Biotec). NALM6 cells were cultivated in RPMI 1640 medium (Biowest) supplemented with 10% fetal bovine serum (FBS) (Biochrom AG) and 2 mM glutamine (Sigma-Aldrich). β2M^−/−, CD47high HEK293T cells were cultivated in DMEM (Gibco) supplemented with 10% FBS and 2 mM glutamine. All cells were cultivated at 37 °C, 5% CO₂, and 90% humidity. Regular testing for mycoplasma was performed on all cell lines using a PCR Mycoplasma Test Kit (PanReac Applichem, Germany).