Comparison of GMP-compliant reagents and stimulation intensity
Small scale studies of EBV T cells cultured in G-Rex flasks comparing reagents and stimulation round densities were performed using frozen vials of matched MNC and LCL supplied from the second-generation SNBTS T cell bank. As before, on day of initiation LCL were irradiated at 40Gy and mixed with autologous MNC at an initial stimulation ratio of 40 MNC : 1 LCL. Cells from each donor were divided and cultured in 40mL culture medium: either RPMI with glutamine + FBS [10%] + research-grade IL-2 [50IU/mL] or xeno-free GMP-grade TexMACS (Miltenyi Biotec) + GMP-grade IL-2 [50IU/mL] (both Miltenyi Biotec) and transferred to G-Rex10cm2 flasks (1x107 MNC + 2.5x105 LCL, final density = 1.0x106MNC per cm2). Cell counts were taken every 3-4 days and cultures re-fed at these time-points by removing 90% volume of media and replacing with fresh media plus IL-2. At day 10, cells were harvested from flasks and re-cultured at 0.5-1x107 per G-Rex10 for re-stimulation with irradiated LCL at 1 T cell : 5 LCL as per standard G-Rex protocol. Cultures in fully GMP reagents were further subdivided into secondary stimulation ratios of 1 T cell : 5 LCL (standard protocol) or 1 T cell : 1 LCL (reduced-intensity modified protocol). A final round of stimulation was performed at day 20, with either 1 T cell : 5 LCL or 1 T cell : 1 LCL for the standard or modified protocol respectively. At day 30, cells were harvested from the G-Rex and assessed for yield and viability by trypan blue exclusion and tested in the flow cytometric assays below.