Generation of EBV-specific T cells in standard culture is improved in G-Rex flasks
Standard cultures were generated by re-deriving new products from frozen stored leukapheresis samples from 6 donors. These new cultures were grown in standard medium (RPMI + 10% FCS and glutamine) in T75cm2 flasks by mixing thawed apheresis MNC with autologous LCL (irradiated at 40Gy) at an initial ratio of 40 MNC : 1 LCL, and then re-stimulated with fresh autologous LCL for a total of 6-8 stimulation rounds. Final T cell product (1.0x107cells/ml) were frozen in cryovials from each stimulation round and stored for later analysis by flow cytometry.
A comparison study set up to assess improvement of culture of EBV-specific T cells using closable G-Rex flasks (Wilson-Wolf) was carried out using thawed donor leukapheresis material and compared against cultures in conventional open culture flasks. Cells in suspension from each donor were split and cultured in either T75cm2 culture flasks (1.0x108 MNC + 2.5x106 LCL, final density = 1.33x106 MNC per cm2) or G-Rex100cm2 flasks (1.0x108 MNC + 2.5x106 LCL, final density = 1.0x106MNC per cm2). At day 10, cells in T75cm2 culture were counted, and a second stimulation of autologous irradiated LCLs were added at a ratio of [4 T cell : 1 LCL] and split to new T75cm2 flasks to give a final density of 0.5-1.0x106 T cells per cm2. For G-Rex cultures at day 10, cells were counted and irradiated LCL added at a ratio of [1 T cell : 5 LCL] and split to new G-Rex100cm2 flasks to give a final density of 0.1x106 T cells per cm2 as indicated in the rapid expansion protocol [14]. At day 14, cultures were re-fed with interleukin-2 (IL-2) at a final concentration of [20IU/mL]. Thereafter, cultures were counted every 3-4 days and fed/split to new flasks as necessary. All cell counts were given as viable cells via trypan blue exclusion of dead cells.