Control of shape and size of vascular smooth muscle cells in vitro by plasma lithography

The ability to control the shape and size of cells is an important enabling technique for investigating influences of geometrical variables on cell ph ysiology. Herein we present a micropatterning technique ("plasma lithograph y") that uses photolithography and plasma thin-film polymerization for the fabrication of cell culture substrates with a cell-adhesive pattern on a ce ll-repellent (non-fouling) background. The micron-level pattern was designe d to isolate individual vascular smooth muscle cells (SMC) on areas with a projected area of between 25 and 3600 mum(2) in order to later study their response to cytokine stimulation in dependence of the cell size and shape a s an indication for the phenotypic state of the cells. Polyethylene terepht halate substrates were first coated with a non-fouling plasma polymer of te traglyme (tetraethylene glycol dimethyl ether). In an organic lift-off proc ess, we then fashioned square- and rectangular-shaped islands of a thin flu orocarbon plasma polymer film of similar to 12-nm thickness. Electron spect roscopy for chemical analysis and secondary ion mass spectroscopy were used to optimize the deposition conditions and characterize the resulting polym ers. Secondary ion mass spectroscopy imaging was used to visualize the spat ial distribution of the polymer components of the micropatterned surfaces. Rat vascular SMC were seeded onto the patterned substrates in serum-free me dium to show that the substrates display the desired properties, and that c ell shape can indeed be controlled. For long-term maintenance of these cell s, the medium was augmented with 10% calf serum after 24 h in culture, and the medium was exchanged every 3 days. After 2 weeks, the cells were still confined to the areas of the adhesive pattern, and when one or more cells s panned more than one island, they did not attach to the intervening tetraet hylene glycol dimethyl ether (tetraglyme) background. Spreading-restricted cells formed a well-ordered actin skeleton, which was most dense along the perimeter of the cells. The shape of the nucleus was also influenced by the pattern geometry. These properties make the patterned substrates suitable for investigating if the phenotypic reversion of SMC can be influenced by c ontrolling the shape and size of SMC in vitro. (C) 2001 John Wiley & Sons, Inc.