Micropatterning of a nanoporous alumina membrane with poly(ethylene glycol) hydrogel to create cellular micropatterns on nanotopographic substrates

In this paper, we describe a simple method for fabricating micropatterned nanoporous substrates that are capable of controlling the spatial positioning of mammalian cells. Micropatterned substrates were prepared by fabricating poly(ethylene glycol) (PEG) hydrogel microstructures on alumina membranes with 200 nm nanopores using photolithography. Because hydrogel precursor solution could infiltrate and become crosslinked within the nanopores, the resultant hydrogel micropatterns were firmly anchored on the substrate without the use of adhesion-promoting monolayers, thereby allow tailoring of the surface properties of unpatterned nanoporous areas. For mammalian cell patterning, arrays of microwells of different dimensions were fabricated. These microwells were composed of hydrophilic PEG hydrogel walls surrounding nanoporous bottoms that were modified with cell-adhesive Arg-Gly-Asp (RGD) peptides. Because the PEG hydrogel was non-adhesive towards proteins and cells, cells adhered selectively and remained viable within the RGD-modified nanoporous regions, thereby creating cellular micropatterns. Although the morphology of cell clusters and the number of cells inside one microwell were dependent on the lateral dimension of the microwells, adhered cells that were in direct contact with nanopores were able to penetrate into the nanopores by small extensions (filopodia) for all the different sizes of microwells evaluated.