Local Polymer Replacement for Neuron Patterning and <i>in Situ</i> Neurite Guidance
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Abstract
By locally dispensing poly-l-lysine (PLL) molecules with
a FluidFM onto a protein and cell resistant poly-l-lysine-<i>graft</i>-polyethylene glycol (PLL-<i>g</i>-PEG) coated
substrate, the antifouling layer can be replaced under the tip aperture
by the cell adhesive PLL. We used this approach for guiding the adhesion
and axonal outgrowth of embryonic hippocampal neurons <i>in situ</i>. Cultures of hippocampal neurons were chosen because they mostly
contain pyramidal neurons. The hippocampus is known to be involved
in memory formation, and the stages of network development are well
characterized, which is an asset to fundamental research. After fabricating
diffuse PLL spots with 10–250 μm diameter, seeded hippocampal
cells stick preferentially onto the spots migrating toward the spot
center along the PLL gradient. Cell clusters were formed depending
on the lateral size of the PLL dots and the density of seeded cells.
In a second step of this protocol, the FluidFM is used to connect <i>in situ</i> the obtained clusters. The outgrowth of neurites,
which are known to grow preferentially on adhesive substrates, is
tailored by writing PLL lines. Antibody staining confirms that the
outgrowing neurites are mostly axons, while the activity of the neurons
is assessed by a calcium indicator, proving cell viability. The calcium
signal intensity of two actively interconnected clusters showed to
be correlated, corroborating the formation of vectored and polarized
interconnections