Emanuele Ostuni

Subcellular positioning of small molecules.

Localized perturbation of processes that take place inside the living cell depends on molecular and spatial discrimination on a micrometre scale. Here we report the use of multiple laminar streams in a microfluidic channel to deliver membrane-permeable molecules to selected subcellular microdomains. This technique opens up avenues for non-invasively visualizing, probing and manipulating the cellular metabolic and structural machinery.

Directional control of lamellipodia extension by constraining cell shape and orienting cell tractional forces

Directed cell migration is critical for tissue morphogenesis and wound healing, but the mechanism of directional control is poorly understood. Here we show that the direction in which cells extend their leading edge can be controlled by constraining cell shape using micrometer‐sized extracellular matrix (ECM) islands. When cultured on square ECM islands in the presence of motility factors, cells preferentially extended lamellipodia, filopodia, and microspikes from their corners. Square cells reoriented their stress fibers and focal adhesions so that tractional forces were concentrated in these corner regions. When cell tension was dissipated, lamellipodia extension ceased. Mechanical interactions between cells and ECM that modulate cytoskeletal tension may therefore play a key role in the control of directional cell motility.—Parker, K. K., Brock, A. L., Brangwynne, C., Mannix, R. J., Wang, N., Ostuni, E., Geisse, N. A., Adams, J. C., Whitesides, G. M., Ingber, D. E. Directional control of lamellipodia extension by constraining cell shape and orienting cell tractional forces. FASEB J. 16, 1195–1204 (2002)

The interaction of proteins and cells with self-assembled monolayers of alkanethiolates on gold and silver

Alkanethiols, HS(CH2)nX, chemisorb on gold and silver and form self-assembled monolayers (SAMs). The ability to present a variety of functional groups, X, at the terminal position of the alkanethiol makes it possible to control the structure of the surface at the molecular level, and thus to control the interfacial properties of these organic surfaces. These SAMs constitute an exceptionally useful set of model surfaces with which to study the interaction of synthetic materials with biologically relevant systems. By varying the terminal group X, it is possible to examine the influence of the structure and polarity of common organic groups on the adsorption of proteins. Alkanethiols terminated with oligo(ethylene glycol) groups form SAMs that resist the adsorption of proteins (so-called ‘inert surfaces’). These alkanethiols, when used in mixed SAMs that include alkanethiols that present other functional groups, isolate the biomolecular interactions of interest from non-specific effects and simplify fundamental studies of protein adsorption. Surface plasmon resonance (SPR) is a particularly valuable technique for measuring rates and equilibrium constants of processes that involve adsorption of proteins at surfaces and for characterizing mechanisms of protein adsorption. Since the techniques used in preparing SAMs for studies of protein adsorption are essentially the same as those used in preparing substrates for SPR, a common synthetic technology can be used with both. Soft lithographic techniques—microprinting and micromolding—make it possible to pattern SAMs with different functionalities on surfaces that can be either planar or contoured. The combination of SAMs, inert surfaces, SPR, and soft lithography allows the study of the molecular-level interaction of solutions containing proteins with synthetic surfaces. Extensions of these studies to investigations of the attachment and spreading of cells on surfaces also offer a new set of research tools in cell biology.

Selective Chemical Treatment of Cellular Microdomains Using Multiple Laminar Streams

There are many experiments in which it would be useful to treat a part of the surface or interior of a cell with a biochemical reagent. It is difficult, however, to achieve subcellular specificity, because small molecules diffuse distances equal to the extent of the cell in seconds. This paper demonstrates experimentally, and analyzes theoretically, the use of multiple laminar fluid streams in microfluidic channels to deliver reagents to, and remove them from, cells with subcellular spatial selectivity. The technique made it possible to label different subpopulations of mitochondria fluorescently, to disrupt selected regions of the cytoskeleton chemically, to dislodge limited areas of cell-substrate adhesions enzymatically, and to observe microcompartmental endocytosis within individual cells. This technique does not require microinjection or immobilization of reagents onto nondiffusive objects; it opens a new window into cell biology.

Topographical Micropatterning of Poly(dimethylsiloxane) Using Laminar Flows of Liquids in Capillaries

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Use of micropatterned adhesive surfaces for control of cell behavior.

Publisher Summary This chapter provides a detailed description of the methods used for the preparation and use of micropatterned surfaces, coated with the extracellular matrix (ECM) protein fibronectin, for analysis of cell behavior using bovine capillary endothelial cells. This approach may be easily adapted for use with other adherent molecules and cell types by adopting appropriate culture medium. The self-assembled monolayer (SAM) and microcontact printing technique presented in this chapter creates adhesive islands surrounded by nonadhesive regions and leads to the adsorption of proteins onto the goldcoated surface in geometrically defined patterns. The use of the microcontact printing technique obviates the need for a dust-controlled laboratory environment after fabrication of the master. This technique also reduces the cost significantly compared to reproducing patterned surfaces for each cover glass using standard photolithographic techniques. It also permits larger scale production of the surfaces, because the photolithographic processing step is only used once during the fabrication process, for the initial fabrication of the master.

Patterning the topographical environment for mammalian cell culture using laminar flows in capillaries

This paper describes the use of patterned flows of multiple laminar streams of etching solutions in capillaries to create various topographical features with sizes of 10-200 /spl mu/m in poly(dimethylsiloxane) (PDMS). A variety of topographical features were created by using channels with obstacles, by adjusting the flow rates of etchant, or by controlling the duration of etching. Bovine capillary endothelial cells aligned parallel to features when grown inside these topographically patterned capillaries with 10 /spl mu/m ridges. The capillaries with topographical features could also be further patterned with surface-attached red blood cells and surface-adsorbed proteins, using laminar flows. This two-stage patterning produces patterns of proteins and cells on the topography with alignment between the different features and patterns. The technique allows simultaneous micropatterning of multiple cell culture environments using the same capillary system.

Soft lithography and surface chemistry: enabling tools for new bioassays

The process of drug discovery can be accelerated by increasing the information content of bioassays and by employing assay platforms that are amenable to high throughput screening techniques. In this paper, we demonstrate how the combination of soft lithography with controlled surface chemistry achieves these goals in a wide spectrum of bioassays. A number of soft lithographic methods can be used to generate micro-structures for the purposes of increasing assay density, diversity of test conditions and improving assay detection qualities. In addition, soft lithography, combined with specific surface chemistry modification procedures and protein engineering, may be used to control the localized molecular and biological properties of substrates, thereby enabling the development of new types of bioassays. The developed methodologies are simple, easily implemented, and lend themselves well to automation. Experimental results and prototypes are presented to illustrate the capabilities of these new techniques. For example, soft lithography and surface chemistry are employed for chemically patterning substrates, stenciling biological entities onto substrates and confining solutions. As a result, information-rich, highdensity bioassays can be obtained where biological targets, surface properties and medium solutions are carefully determined and controlled.