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