In vitro T cell activation and lentivirus transduction Grex vessels (WilsonWolf) and Permalife cell culture bags (Origen Biomedical)
T cells or leuko-apheresed cells were stimulated in vitro with ImmunoCult CD3/28/2 activator or Immunocult CD3/28 activator at 25 uL per 1 mL of cells in complete culture media for 16-24 hours before transduction. Prior to transduction, viable cell counts were determined using NC-200. Amount of lentivirus at MOI=1 according to their functional titer were used in all experiments except for the lentivirus titration studies. 24 hours following transduction, culture media were topped to 5 mL in small-scale Grex experiments.
To activate and transduce T cells in Permalife cell culture bags, Permalife cell culture bags were coated with OKT CD3 antibody (Milteniyi Biotech) overnight at 4°C prior to cell inoculation. Enriched T cells were transferred to Permalife cell culture bags with soluble 15E8 CD28 antibody (Milteniyi Biotech) followed by transduction at MOI=1 24 hours after activation. Cells were transferred into Xuri W25 bioreactor 1 day after transduction for scale-up expansion.