A method for patterning multiple types of cells by using electrochemical desorption of self-assembled monolayers within microfluidic channels.

This report describes a method for patterning multiple types of adherent cells on the same substrate by electrochemical desorption of self-assembled monolayers (SAMs) in localized areas defined by a microfluidic system. Several groups have previously reported techniques that allow the patterning of two different types of cells. None of these reported techniques, however, could both confine two or more types of cells to specific locations on surfaces without the presence of physical constraints and control the motility of these different types of cells. 4, 5] The technique presented herein will be useful for a number of biological systems, such as in the studies of neuronal development and in the control of tumor growth. Our method employs a commercially available thiol (HS(CH2)11(OCH2CH2)6OH, abbreviated as “EG6”) to form a SAM on the gold surface, which resists adsorption of proteins and adhesion of cells (for convenience, we call this surface the “inert surface”). A poly(dimethylsiloxane) (PDMS) stamp with embedded microfluidic channels is used to carry out selective electrochemical desorption of EG6 from the gold substrate (Figure 1). [7] This procedure allows parts of the inert surface to promote the adsorption of proteins and the adhesion of cells (we call this transformation “activation of the inert surface”). Each of these individually addressable microchannels can deliver one type of cell to activated regions of the surface, resulting in a pattern of multiple types of cells on the surface. Because an electrochemical reaction can take place only in areas exposed to microfluidic channels, patterned cells are confined to activated regions, which are defined by these channels upon removal of the stamp that carries the fluidic system. As there is no physical barrier between these cells, there is a free exchange of substances between these different types of cells through the liquid medium. This exchange allows the studies of cell–cell interactions when different types of cells are confined to separate locations on the surface. A second step of electrochemical desorption can “turn on” motility of cells and allow them to move under the influence of each other. We illustrate this approach by patterning two types of cells (NIH 3T3 and Hela cells) in stripes. Fabrication of an inert surface is accomplished by coating a gold-covered glass substrate with EG6. To selectively activate the inert surface, we first coated the inert substrate with a PDMS stamp with embedded microfeatures (see the Supporting Information for its fabrication) to form enclosed microchannels. The features embedded in the PDMS stamp formed the ceilings and vertical walls and the gold substrate formed the floors of the channels; the channels were reversibly sealed. We filled the channels with solutions of the extracellular matrix (ECM) protein fibronectin (100 mgmL 1 in a phosphate-buffered Figure 1. Strategy for patterning different types of cells. a) To obtain “inert” surfaces, we formed SAMs on gold-coated coverslips with EG6. b) A PDMS stamp with an embedded microfluidic system was brought into contact with the substrate, and the channels were filled with solutions of fibronectin. Application of a cathodic potential on the gold substrates desorbed SAMs inside the channels. c)–f) Magnified views of the main functional locations of the channel system. c) Adsorption of proteins inside the microchannels after electrochemical activation of the surface. d) Adhesion of cells on the floors of the channels. e) After the PDMS stamp was peeled off, a pattern of different types of cells was formed. f) A second step of electrochemical desorption enabled cells that were previously confined in patterns to spread across the previously inert surface.