Cellular micropatterning has become an important tool to precisely design cell-to-substrate attachment for cell biology studies, tissue engineering, cell-based biosensors, biological assays, and drug screens. This paper describes a new technique for micropatterning of cells that is based on the use of oxygen plasma as a patterning tool. The technique consists of (1) homogeneously grafting a glass substrate with a protein-repellent interpenetrating polymeric network (IPN) of poly(acrylamide) and poly(ethyleneglycol) [P(AAm-co-EG)] prepared with commercially available reagents and (2) selectively removing this coating using oxygen plasma. We use elastomeric stencils (i.e. self-sealing membranes with through-holes) and microchannels as removable masks for the selective oxygen plasma etch of the IPN areas that are not protected by the mask. The stencil or microchannels are peeled off to reveal cell-adhesive regions separated by the nonadhesive coating. Our method offers a convenient way of patterning a robust protein-repelling material, allows for independently controlling the chemistry of the regions reserved for cell attachment, and can be used to create coculture systems.

The ability to produce three-dimensional (3D) microstructures is of increasing importance in the miniaturization of mechanical or fluidic devices, optical elements, self-assembling components, and tissue-engineering scaffolds, among others. Traditional photolithography, the most widely used process for microdevice fabrication, is ill-suited for 3D fabrication, because it is based on the illumination of a photosensitive layer through a ''photomask'' (a transparent plate that contains opaque, unalterable solid-state features), which inevitably results in features of uniform height. We have devised photomasks in which the light-absorbing features are made of fluids. Unlike in conventional photomasks, the opacity of the photomask features can be tailored to an arbitrary number of gray-scale levels, and their spatial pattern can be reconfigured in the time scale of seconds. Here we demonstrate the inexpensive fabrication of photoresist patterns that contain features of multiple and͞or smoothly varying heights. For a given microfluidic photomask, the developed photoresist pattern can be predicted as a function of the dye concentrations and photomask dimensions. For selected applications, microfluidic photomasks offer a low-cost alternative to present gray-scale photolithography approaches. Photolithography is used to define critical feature size in the fabrication of the vast majority of microdevices including microelectronic circuits, microelectromechanical systems (MEMS), microfluidic devices, and nucleic acid͞protein microarrays (1). Essentially, photolithography consists of selectively illuminating a thin photosensitive layer (''photoresist'') with UV light through a mask containing opaque features (e.g., metal or ink emulsion) on a transparent background (e.g., glass or plastic). The photomasks impose two fundamental limitations on the features that can be produced. (i) The exposure is an all-or-none illumination process that results in photoresist features of uniform height; thus, the fabrication of threedimensional (3D) microstructures by traditional photolithography requires multiple exposure͞alignment steps. (ii) Photomask features are permanent, and thus design changes require the (costly and slow) fabrication of a new photomask, a major hurdle in research settings requiring fast-turnaround microdevice prototyping.To overcome the all-or-none illumination constraint of conventional photolithography, a number of ''gray-scale'' approaches capable of generating ranges of exposure levels have been developed. Presently, gray-scale photolithography can be realized with scanning lasers (2, 3), micromirror projection displays (4), high-energy beam-sensitive glass photomasks (www. canyonmaterials.com), ultra-high-resolution ''halftone'' photomasks (5), or metal-on-glass photomasks [where each gray-scale level is determined by a different metal thickness (6)]. Although extremely useful for producing 3D structures, these gray-scale approaches either (i) exacerbate the costs͞turnaround times of standard photolithography ...