6.3 Cell Culture Conditions
Certain ingredients, such as electrolytes and macromolecules, and the pH of the cell
culture medium may alter the surface and physico-chemical properties of the NPs
and, thereby influence the gene delivery efficiency. In particular, presence of serum
or serum proteins is often reported to lower the transfection efficiencies of several
NPs. Even if the NPs are stable in the presence of serum or serum proteins, batch
variations in serum quality can lead to differences in transfection efficiency. Thus,
it is often advisable to test a small lot of serum from a reputable supplier in a control
experiment. Once a given lot has yielded satisfactory and reproducible results, sera
from this lot should be used for further experiments.
Cell culture substrate can also the influence gene delivery by affecting the
occurrence of endocytosis and, thereby, the uptake of DNA-bound NPs by the
cells. Hsu et al. [139] observed that the culture of MSCs on chitosan or HAmodified chitosan membranes increased the intracellular uptake of iron oxide NPs
(~5 nm) as well as naked DNA (3.3 kb, ~5 nm) by more than fivefold. The increased
internalization of NPs was associated with an increase in clathrin-mediated endocytosis on chitosan (~50%) and in caveolae-mediated endocytosis on chitosan-HA
(~30–40%). In the case of naked DNA, but not iron oxide NPs, macropinocytosis
also occurred on both substrates.
Microbial contamination by bacteria, fungi and mycoplasma during in vitro cell
culture are additional factors that may influence the efficiency of gene transfection
by modifying the growth behaviour of the infected cells. Variation in the growth
behaviour of infected cells may lead to different transfection efficiencies between
replicate experiments.
6.4 Cell Density and Passaging
In most cases, the optimal confluency for gene transfection for adherent monolayer
cells is ~40–80%. If cell density, at the time of adding NP–DNA complexes, is not
optimal, it can lead to insufficient uptake of complexes into the cells. Furthermore,
cells that have been passaged a large number of times tend to change their growth
behaviour, morphology and potential for transfection. When cells with high passage
numbers are used for replicate experiments, decreased transfection efficiencies may
be observed in later experiments. Using cells with a low passage number (<50
splitting cycles) is, therefore, recommended.
6.5 DNA (Vector Design) Quality and Quantity
The DNA backbone of a vector as such does not influence the gene delivery efficiency
of NPs. However, the type of promoter, regulatory sequences, coding sequence, etc.
74
P. Pushp et al.
Certain ingredients, such as electrolytes and macromolecules, and the pH of the cell
culture medium may alter the surface and physico-chemical properties of the NPs
and, thereby influence the gene delivery efficiency. In particular, presence of serum
or serum proteins is often reported to lower the transfection efficiencies of several
NPs. Even if the NPs are stable in the presence of serum or serum proteins, batch
variations in serum quality can lead to differences in transfection efficiency. Thus,
it is often advisable to test a small lot of serum from a reputable supplier in a control
experiment. Once a given lot has yielded satisfactory and reproducible results, sera
from this lot should be used for further experiments.
Cell culture substrate can also the influence gene delivery by affecting the
occurrence of endocytosis and, thereby, the uptake of DNA-bound NPs by the
cells. Hsu et al. [139] observed that the culture of MSCs on chitosan or HAmodified chitosan membranes increased the intracellular uptake of iron oxide NPs
(~5 nm) as well as naked DNA (3.3 kb, ~5 nm) by more than fivefold. The increased
internalization of NPs was associated with an increase in clathrin-mediated endocytosis on chitosan (~50%) and in caveolae-mediated endocytosis on chitosan-HA
(~30–40%). In the case of naked DNA, but not iron oxide NPs, macropinocytosis
also occurred on both substrates.
Microbial contamination by bacteria, fungi and mycoplasma during in vitro cell
culture are additional factors that may influence the efficiency of gene transfection
by modifying the growth behaviour of the infected cells. Variation in the growth
behaviour of infected cells may lead to different transfection efficiencies between
replicate experiments.
6.4 Cell Density and Passaging
In most cases, the optimal confluency for gene transfection for adherent monolayer
cells is ~40–80%. If cell density, at the time of adding NP–DNA complexes, is not
optimal, it can lead to insufficient uptake of complexes into the cells. Furthermore,
cells that have been passaged a large number of times tend to change their growth
behaviour, morphology and potential for transfection. When cells with high passage
numbers are used for replicate experiments, decreased transfection efficiencies may
be observed in later experiments. Using cells with a low passage number (<50
splitting cycles) is, therefore, recommended.
6.5 DNA (Vector Design) Quality and Quantity
The DNA backbone of a vector as such does not influence the gene delivery efficiency
of NPs. However, the type of promoter, regulatory sequences, coding sequence, etc.
74
P. Pushp et al.