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.