In order to do cellular patterning, I first had to find an appropriate chemistry to exclude the cells from the surface, making sure they could only stick where I desired. To that end, we used chemistry from the Rubner lab at MIT, a series of polyelectroylyte layers, as described in the figure on the right. Essentially, using electrostatic interaction at acidic pH, the layers are deposited on a glass surface, then cross-linked. Once thermally cross-linked, the layers are stable. When the sample is subsequently immersed in aqueous solution, the layers swell. Swelled, extremely hydrophilic layers seem to have a cell-exclusive effect (PEG is the classic literature example of this), though the mechanism is poorly understood. Parts of the surface are then modified, through the application of soft lithography, involving a chemically active “ink” on a PDMS “stamp”. The areas stamped are then treated to cause cell-adhesive protein or peptides (fibronectin, RGD) to be deposited in those areas only. When cells are seeded, they assemble on these patterned areas.
Pictured above is an example of this technique – these are IC-21 (mouse macrophage) cells fixed and stained with Hoechst 33342 (blue: nuclear stain), CMFDA (green: cell volume), and Texas Red Phalloidin (Red: actin). It is realatively easy to use soft lithography at this scale, with 10 μm patterns working flawlessly.
On the left is an example of a smaller pattern, 2 x 2 μm squares with 1 μm spacing between them. This pattern was slightly harder to achieve, as smearing of the pattern is easy at this size scale. This pattern is made of stained protein, used to visualize the location of the adhesive areas.