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First published online December 1, 2003
doi: 10.1242/10.1242/jcs.00836

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Cell behaviour on micropatterned substrata: limits of extracellular matrix geometry for spreading and adhesion

¹ Department of Biology, University of Konstanz, Universitaetstrasse 10, 78457 Konstanz, Germany
² Department of Pathology, Centre Medical Universitaire, Geneva, Switzerland
³ Laboratory for Micro- and Nanotechnology, Paul Scherrer Institut, Villigen-PSI, Switzerland

View larger version (30K): [in a new window]   Fig. 1. Schematic of the microcontact printing method. (A) A PDMS stamp is prepared from a silicon master structure and `inked' with a hydrophobic alkanethiol. The thiol pattern is stamped onto a gold-coated coverslip, forming self-assembled monolayers. The remaining regions are blocked with a protein-resistant hydrophilic alkanethiol. ECM proteins now selectively adsorb to the hydrophobic areas of the coverslip. (B) The PDMS stamp used in this study consists of 2x9 fields, each containing a unique arrangement of squared dots varying in size and distance (a, length of dots in µm; d, centre to centre distance of dots in µm). The theoretical protein coverage for each field is given as a percentage.

View larger version (54K): [in a new window]   Fig. 2. Protein adsorption on micropatterned substrata. Scanning force microscopy of a micropatterned substratum coated with fibronectin (dots of 1.2 µm square at a distance of 5 µm). (A) Low magnification scan (scan rate 0.5 Hz, total z scale 15 nm) showing that fibronectin is almost exclusively adsorbed to the hydrophobic alkanethiol dots. (B) Higher magnification scan of a fibronectin-coated dot. (C) Bearing histogram of the boxed region in B reveals a mean height difference between the fibronectin-coated area and the surrounding surface of approximately 3 nm. (D) Height profile (line and arrowheads in B; 3x3 µm2 scan, scan rate 1.5 Hz) showing that fibronectin is adsorbed mainly as a monolayer, but up to three layers can be measured.

View larger version (90K): [in a new window]   Fig. 3. Cell spreading on micropatterned substrata. A B16 mouse melanoma cell cultured for 1 hour on a patterned substratum of fibronectin. (A) Overlay of fibronectin immunofluorescence (white dots: 1 µm2; dot distance centre to centre: 5 µm) on a differential interference contrast image. The substratum determines the cell shape, resulting in a rectangular morphology. (B) Overlay of the fibronectin pattern and phalloidin staining reveals that most actin fibres terminate at fibronectin dots. Scale bar: 10 µm.

View larger version (48K): [in a new window]   Fig. 4. Molecular composition of focal contacts on micropatterned substrata. B16 cells were cultured for 1 hour on patterned ECM substrata and labelled for focal adhesion molecules and ECM proteins. (A,A') Cell on a homogenous fibronectin substratum prepared with µCP. Fluorescence staining for vinculin (Vin, green) and actin (Act, red) reveals a staining pattern of dot-like or elongated adhesion foci mainly at the cell periphery that were connected to actin bundles. (B,B') Cell stained for vinculin (Vin, green) and actin (red) on a patterned substratum of 0.6 µm2 fibronectin dots (FN, blue). Vinculin has accumulated in areas of the cell overlying ECM dots. Actin fibres terminate in most of these adhesion sites, indicating functional contact sites. (C,C') Cell stained for focal adhesion kinase (FAK, green) on a patterned substratum of 1 µm2 FN dots (red). (D,D') Cell stained for phosphotyrosine (PT, green) on a patterned substratum of 1 µm2 FN dots (red). (E,E',E'') B16 cell expressing ß3-integrin-GFP (green) stained for paxillin (Pax, red) on a patterned vitronectin (VN, blue) substratum of 0.6 µm2 dots. (F,F') B16 cell expressing ß 3-integrin-GFP (green) labelled for actin (red) growing on vitronectin (blue) at the border between a uniform and a patterned substratum of 1 µm2 dots. Note the redistribution of integrin receptors on the patterned substratum. Scale bars: 10 µm.

View larger version (105K): [in a new window]   Fig. 5. Cell spreading on polylysine. B16 cells were cultured on patterned substrata of fibronectin (red; A) or polylysine (red; B,C) and labelled for actin (green; A,B) and paxillin (green; C). Cells can adhere to polylysine dots, however, the actin cytoskeleton is not reorganised (B) and paxillin does not accumulate over the dots (C). Scale bars: 10 µm.

View larger version (116K): [in a new window]   Fig. 6. Cell spreading in relation to substratum geometry. B16 cells were cultured on fibronectin substrata prepared with µCP and labelled for fibronectin (red) and actin (green). (A) On a homogeneous substratum (hs), actin filaments are distributed throughout the cell periphery. (B,C) If the space between dots is 2 µm (B: 0.1 µm2 squares 1 µm apart, C: 1 µm2 squares 2 µm apart) cells spread as on a homogeneous substratum. (D-I) Cell growth on patterned substrata of 9 µm2 dots with spacing as indicated in the right-hand corner. (D-F) With distances of 5-20 µm between dots, cells spread and the actin cytoskeleton formed stress fibres between adjacent dots. (G-I) At a distance of 25 µm, spreading was limited and cells became triangular, ellipsoid or round. Scale bar: 10 µm.

View larger version (93K): [in a new window]   Fig. 7. Fibronectin dots of 0.1 µm2 induce intracellular signalling but do not support cell spreading at distances >5 µm. (A) B16 cells growing on patterned fibronectin substrata with varying dot sizes (as indicated in the lower left corner) and a constant spacing of 5 µm (centre to centre). Cells spread and form actin stress fibres on dots down to 0.25 µm2. On 0.1 µm2 dots, cells adhere but do not spread. Note missing dots in the vicinity of the cells on 0.25 µm2 and 0.1 µm2 substrata. Scale bar: 10 µm. (B) A cell sitting on 0.1 µm2 dots at a spacing of 2 µm. Phosphotyrosine (PT, green) accumulates in areas of the cell overlying fibronectin dots (red) indicating that an area of 0.1 µm2 is sufficient to induce intracellular signalling. (C) Staining for paxilin (Pax, green) of a cell sitting on 0.1 µm2 fibronectin dots (red) separated by distances of 4 µm. Like phosphotyrosine, paxilin accumulates over small dots. Scale bars: 5 µm.

View larger version (121K): [in a new window]   Fig. 8. Dynamics of cell spreading on fibronectin dots of 0.1 µm2. Images of a time-lapse sequence of a B16 cell expressing ß3-integrin-GFP (ß3-GFP) growing on a patterned substratum of 0.1 µm2 fibronectin dots. Dots were visualised by mixing the fibronectin with fluorescently labelled BSA. Since ß3-GFP cells display homogeneous background fluorescence, they were used in these assays to reveal cell morphology. The cell was highly motile but did not spread. A few minutes after initial contact, a dot (arrows in A-F) was first stretched (arrow in C), removed from the substratum (arrowhead in D) and then internalised into the cell (arrowheads in E and F). This behaviour results in a rearranged dot pattern after a cell has migrated over that area. Time is given in minutes and seconds. Scale bar: 10 µm.

View larger version (23K): [in a new window]   Fig. 9. Correlation between cell spreading and substratum coating. (A,B) The area covered by NIH/3T3 cells (A) and B16 cells (B) that have spread was plotted against the actual fibronectin surface for 12 different dot patterns, uncoated and homogeneously coated substrata. Bars represent s.e.m. (C) Three representative cells from patterns with 4% fibronectin coverage (arrow in B). Cell sizes as indicated in the upper right-hand corner were almost equal despite the different fibronectin patterns (actual length and margin to margin distance of dots is given in µm in the upper left corner).

View larger version (36K): [in a new window]   Fig. 10. Quantification of dot usage. B16 cells were cultured for 1 hour on patterned substrata and labelled for fibronectin (FN), actin (act) and phosphotyrosine (PT) or paxillin (Pax). The area covered by phosphotyrosine (A) or paxillin (B) was measured for focal adhesions in the cellular periphery for cells growing on homogeneous substrata (hs) and on patterned substrata with three different dot sizes (contact size, ) and set in relation to the underlying fibronectin area (dot usage, ). Dot usage was also determined for focal adhesions formed on patterned substrata with 12 µm2 dots and variable distances. Bars represent s.e.m.

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