(596as) Calcium-Alginate Mediated Nucleic Acid Delivery in a Microfluidic Device

Calcium alginate entrapment
of cells has been used extensively in cell culture for cellular and tissue
engineering applications. We recently described a method to co-deposit and
culture cells inside a calcium alginate hydrogel by means of a DC electric
signal [1] that also allows for the study of intercellular signaling phenomena
in a microfluidic device [2]. We report an advance that enables the
site-specific genetic reprogramming of cells- introducing a plasmid into the
cells by electrically-assisted chemical transformation as they are entrapped
within the calcium alginate hydrogel. In addition, this system allows for
direct optical access to the cells throughout the entire hydrogel, giving
researchers a new level of control over cellular engineering experiments  and the delivery of nucleic acids to cell
populations as well as the ability to study spatial distribution, migration,
and responses of reprogrammed cells through the use of confocal microscopy.

A mixture of 1% (w/v) sodium alginate solution and
0.5% (w/v) CaCO₃ nanoparticles was electrodeposited at a constant
current density of 10 A/m² for 30 seconds at each of four electrodes
inside a microfluidic device whose fabrication is described previously [2]. A mixture of 0.5% (w/v)
sodium alginate solution and 20 ng/µL plasmid was
mixed gently with an equal volume of electrocompetent
Escherichia coli cells and
electrodeposited at a constant current density of 6 A/m² for 3
minutes at the same electrodes. The transformed cells were then allowed to
recover at 37ºC for an hour in LB media before flowing 1 µL/min LB selection
media supplemented with appropriate antibiotics and 40 mM
CaCl₂ to maintain hydrogel integrity. Induction media, which is
identical to the selection media except for the addition of 1 mM IPTG, was added at 1 µL/min after bacteria reached
mid-log phase for experiments requiring induction.

The deposition of a small calcium alginate hydrogel
containing an excess of CaCO₃ enables the electrodeposition of a
calcium alginate hydrogel that is free of CaCO₃ particles, ensuring
optical access to the cells throughout the entire hydrogel that was observed by
transmitted light (Fig 1a) or fluorescence (Fig 1b). An optical density of the
cells in the hydrogel was calculated by transmitted light (Fig 1c) and fluorescent
protein production by the cells was monitored by fluorescence microscopy (Fig
1d). Confocal microscopy demonstrates this method is superior to previous methods
[1, 2] by allowing optical access through the entirety of the hydrogel rather
than the ~100 µm closest to the objective lens. Control experiments indicate
cells are transformed by a synergistic combination of electric field, Ca²⁺,
and Joule heating. IPTG can be used to induce protein expression at a desired
time point, as demonstrated in Figure 2. Transformed cells were cultured and
used to produce protein constitutively or selectively induced by environmental
factors, effectively acting as sensors. The most exciting aspect of this
simple, rapid, and reagentless technique is the
site-selective reprogramming of cells as they are entrapped within a calcium
alginate hydrogel, offering researchers both spatial and temporal control over
the genetic makeup of cell populations within the cell culture environment.

E. coli cells have been genetically reprogrammed by an electric
field-assisted chemical transformation mechanism while they are simultaneously
entrapped and cultured within a calcium alginate hydrogel at an electrode
surface. This process enables selective transformation of desired cell
populations at a specific spatial location. Cell growth and fluorescent protein
expression were easily monitored optically within the microfluidic device.
Transformed E. coli can act as
cell-based sensors of chemicals in the environment. This powerful and versatile
method is currently being extended into the study of intercellular
communication phenomena and is being adapted for eukaryotic cell lines such as
Sf9 and HEK293, which are important to the biomedical engineering field for
basic research, vaccine development, and therapeutic protein production.

Acknowledgements: This work was supported by a grant from the
Robert W. Deutsch Foundation.

References:

1)     
Shi, X.-W., et al., Adv.
Funct. Mater., 2009,  19, 2074-2080.

2)     
Cheng, Y., et al., Adv.
Funct. Mater., 2012,  22, 519-528.

Figure
1. (a) Transmitted light image of E. coli entrapped in a calcium alginate hydrogel. (b) False color fluorescent
image of DsRed expressed by E. coli in (a). (c) Optical density measured from transmitted light
images of hydrogels over 24 hour period. (d) DsRed
expression measured by fluorescence images of hydrogels over 24 hour period.

Figure 2. IPTG induction of DsRed
expression in E. coli transformed
during electrodeposition.  Protein
expression is minimal until IPTG induction begins at 10 hours post
transformation, at which point it begins to increase at a rate much higher than
previously measured.