Lipofection of Primary T Cells
After determining the optimum vehicle (Lipofectamine LTX), promoter (EF1α), and media formulation (serum-free X-VIVO) in Jurkat cells, we used the same conditions to transfect CD3+ primary T cells. Surprisingly, this protocol yielded much lower transfection efficiencies in the primary T cells (8.1±0.8% EGFP+) than the Jurkat T cells (Figure 4A). This dramatic decrease in transgene expression is also illustrated by the histograms in Figures 4C/D and was visually apparent when observing the cells with fluorescent microscopy, since the Jurkat cells fluoresced brightly while the primary T cells were relatively dim. It is worth noting that primary T cells are cultured with additional components (recombinant IL-2 and anti-CD3/CD28 Dynabeads) that could potentially interfere with transfection, but culturing Jurkat T cells with Dynabeads and IL-2 did not significantly decrease transfection efficiency (data not shown).
A previous study using cationic pHEMA-g-pDMAEMA polymers observed a similar disparity, in which the polymer provided 50% transfection efficiency in Jurkat T cells but only 18-25% transfection efficiency in CD4+ and CD8+ primary T cells.45 This same group later showed that endosomes in primary T cells acidify at a slower rate than endosomes in HeLa cells, which could slow the rate of endosomal escape for non-viral gene delivery vehicles that rely upon the proton sponge effect to destabilize the endosome and escape into the cytoplasm.73
Another variable that significantly influence transfection efficiency in primary T cells was the timing (0-3 days) between activation and transfection (Figure 4B). Indeed, other groups have previously shown that the time between these two steps significantly impacts gene delivery with viruses, electroporation, and cationic polymers.25,45,12–14 In our experiments, cells were either transfected 30 minutes after the addition of anti-CD3/28 Dynabeads to the cells (i.e., Day 0) or 24, 48, and 72 hours later (i.e., Days 1-3). Interestingly, transfection efficiency was highest when lipoplexes were added to the cells on Day 0 or Day 1, but decreased significantly on Days 2 and 3. This downward trend in transfection may be due to the fact that cell size and substrate uptake are both maximized immediately after activation.74,75Additionally, cell division spikes after activation, which would allow for more effective entry into the nucleus compared to cells that are in a resting state.76