Figure 2. Production of AAV6 via triple transient transfection at medium cell densities. Cells were transfected at low cell density (LCD – 1 × 106 cells/mL) as control or at medium cell density (MCD – 4 × 106 cells/mL). Plasmid DNA was delivered on a volumetric basis (VB - 1 µg/mL) or cell basis (CB - 1 µg/106 cells). A) Cell growth kinetics and viability. B) Volumetric (VG/mL) and cell-specific viral yields (VG/cell). Time expressed in hours post-transfection (hpt). C) Transfection efficiency is described as the percentage of cells expressing the transgene (GFP) 24 hpt D) Functional titer (enhanced transducing units (ETU)/mL). E) Ratio between the genomic titer and functional titer (VG/ETU) 48 hpt. Values represent mean ± standard deviation (n=2). **** P < 0.0001, *** P < 0.001, by ANOVA followed by Dunnett’s test and Student’s T-test.