Micropatterns of Chemisorbed Cell Adhesion-Repellent Films Using Oxygen Plasma Etching and Elastomeric Masks

Abstract

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.

Biology on a Chip: Microfabrication for Studying the Behavior of Cultured Cells

“…We cultured myogenic cells (C2C12 cell line) on microtracks of physisorbed fibronectin or Matrigel™ surrounded by protein-repellent PEG IPN. 28 PEG IPNs repelled cell adhesion and spread robustly over a long term (>4 weeks; we have not determined the upper value). To produce cell-adhesive tracks alternating with IPNs, we first homogeneously grafted PEG IPNs onto glass substrates and then used PDMS microchannels to selectively remove IPNs with oxygen plasma and to deposit ECM proteins onto etched tracks.…”

“…The pattern was created by selective etching of a homogeneous PEG IPN layer through a PDMS stencil. 28 When constrained to this pattern for long periods of time (Fig. 5C, 21 days), the neurons will eventually reach to neighboring islands and join their axons in bundles.…”

“…26 Figure 3G depicts stencil patterning, whereby a PDMS membrane is fabricated by exclusion of PDMS from the top of the photoresist master features, resulting in holes with the shape of the master features. PDMS can be excluded from the top of the features by (1) pressing a plate against the master after the PDMS has been poured over the master, 27 (2) capping the dry master with a cover (which effectively forms a microfluidic chamber) and using suction or injection to fill the space between the cover and the master, 27,28 or (3) spinning a PDMS prepolymer on the master with photoresist posts to a thickness smaller than the height of the posts. 29,30 The stencil can be used as a mask for selective adsorption of cell-adhesive proteins to promote selective cell attachment (Fig.…”

“…Microfabricated devices have been developed to measure human red blood cell deformability using a single micropore (diameters down to 1 μm) on a thin (0.4 μm thick) Si 3 N 4 film, 282 using 2.5-4-μm-wide microchannels on a silicon wafer 283,28 or PDMS channels. [285][286][287] Bitensky's group 285,286 used an in vitro model of the blood microcirculatory system consisting of a network of PDMS microchannels patterned after the dimensions and architecture of the mammalian microcirculation.…”

Patterned cell culture inside microfluidic devices

“…Recently, Tourovskaia et al have reported a method for generating cellular patterns on substrates coated with interpenetrating polymeric network (IPN) of poly(acrylamide) and poly(ethyleneglycol) film by patterned etching with oxygen plasma. 18 Although this method was successful in generating cell-adhesive areas by removing cell non-adhesive IPN film (19 nm thick), 19 it required approximately 15 min of repeated exposure to plasma. This limited the smallest feature to 15 mm because the elastomeric mask was heated and distorted during plasma treatment.…”