Micropatterned multicolor dynamically adhesive substrates to control cell adhesion and multicellular organization

Abstract

We present a novel technique to examine cell-cell interactions and directed cell migration using micropatterned substrates of three distinct regions: an adhesive region, a nonadhesive region, and a dynamically adhesive region switched by addition of a soluble factor to the medium. Combining microcontact printing with avidin-biotin capture chemistry, we pattern nonadhesive regions of avidin that become adhesive through the capture of biotinylated fibronectin. Our strategy overcomes several limitations of current two-color dynamically adhesive substrates by incorporating a third, permanently nonadhesive region. Having three spatially and functionally distinct regions allows for the realization of more complex configurations of cellular cocultures as well as intricate interface geometries between two cell populations for diverse heterotypic cell-cell interaction studies. We can now achieve spatial control over the path and direction of migration in addition to temporal control of the onset of migration, enabling studies that better recapitulate coordinated multicellular migration and organization in vitro. We confirm that cellular behavior is unaltered on captured biotinylated fibronectin as compared to printed fibronectin by examining the cells' ability to spread, form adhesions, and migrate. We demonstrate the versatility of this approach in studies of migration and cellular cocultures, and further highlight its utility by probing Notch-Delta juxtacrine signaling at a patterned interface.

Figure 1

Generating three-color dynamically adhesive substrates via two microcontact printing techniques. (a) (i) Forward printing. (1) Transfer the fibronectin (red) on a previously inked stamp to the cell culture substrate. (2) Then, transfer the Neutravidin (green) on a previously inked stamp to the same cell culture substrate by manually aligning features as needed. (3) Finally, incubate the substrate in 0.2% Pluronics F127 (w/v) in water for 1 h to render the remaining regions nonadhesive. The fluorescent light (FL) micrograph shows an example of corresponding features. (ii) Stamp-off. (1) Use a UV ozone-activated template to stamp off undesired regions of fibronectin (red) from a previously inked stamp. (2) Re-ink the stamp with Neutravidin (green). (3) Finally, transfer the fibronectin–Neutravidin pattern on the stamp to the cell culture substrate. The fluorescent light (FL) micrograph shows an example of corresponding features. (b) Switch mechanism. Neutravidin patterned regions are nonadhesive to cells but will capture biotinylated fibronectin in solution to then become adhesive. The fluorescent light (FL) micrograph shows an example of corresponding features from (a i, ii) where biotinylated fibronectin labeled with AlexaFluor-647 attaches specifically to the Neutravidin regions and not the fibronectin regions (red) or the nonadhesive regions (black). All scale bars, 100 μm.

Figure 2

Characterization of cellular behavior on dynamically adhesive substrates. (a) Cell spread area is shown and (b) computed from HUVECs seeded on the indicated matrix for 24 h, fixing and immunolabeling for F-actin. (c) Number of focal adhesions are shown and (d) computed from HUVECs seeded on the indicated matrix for 24 h, fixing and immunolabeling for vinculin. (e) HUVECs were followed via time-lapse phase microscopy on the indicated substrates for 2–4 h. Migration tracks, and mean squared displacement versus time was determined and fit to the persistent random walk model to describe cell migration. (f) The parameters speed and persistence time were computed from the model. Box and whisker plots are 5–95%. Scale bars, 25 μm.

Figure 3

Patterning cellular migration. (a) Phase contrast micrographs of HUVECs initially patterned on 35 μm × 35 μm printed fibronectin squares for 12 h, and after the addition of biotinylated fibronectin to the culture to permit cell migration. Scale bars, 100 μm. (b) Migration tracks were recorded from phase contrast images taken every 3 min, for 24 min before addition of biotinyated fibronectin (blue lines), and 48 min after addition of biotinylated fibronectin (red lines). Scale bar, 10 μm. (c) The distance from the initial point over time was computed. Individual cell curves are shown in gray, and the mean, and mean ± sem of cells shown in the plot are shown in solid and dashed red curves, respectively. (d) Schematic showing the technique to pattern cellular migration. In a separate experiment from parts a–c, cells were seeded on a three-color dynamically adhesive substrate. (i) Cells attached only onto fibronectin regions (red) (ii). Biotinylated fibronectin was then added to the media, and cells were free to migrate onto Neutravidin regions only (iii), thus restricted to predefined tracks. Scale bars, 100 μm. Ellipses were fitted to cells before and after adding biotinylated fibronectin, and the major/minor axis length was computed (iv). The box and whisker plot shows the 5–95% range, and the dotted line represents the major/minor axis ratio expected of a perfect circle (major axis/minor axis = 1).

Figure 4

Patterning cellular cocultures. (a) Schematic showing the technique to pattern cellular cocultures. One population of cells is initially seeded on a three-color dynamically adhesive substrate and can only attach to patterned regions of fibronectin (red) and not onto Neutravidin regions (green) or nonadhesive regions (black). After the first cell population fills the fibronectin region completely (cells are cultured for 24 h in serum-free media), biotinylated fibronectin (cyan) is then added to the media. The second population of cells is immediately seeded, and can attach to the “switched” Neutravidin regions but not the nonadhesive regions (black). (b) Top panel: A fibronectin triangle (red) patterned adjacent to a Neutravidin triangle (green). Bottom panel: A single cell (MSC labeled with CellTracker Red) was initially seeded and was only able to attach to the fibronectin region. Biotinylated fibronectin was added to the media, and a second cell type (MSC labeled with CellTracker Green) was then able to attach to the “switched” Neutravidin region, thereby generating a patterned coculture of heterotypic cell pairs. (c) Top panel: Single cell-wide lines of Neutravidin (green) are patterned perpendicular to a single cell-wide line of fibronectin (red). Bottom panel: Two separate cell types (Notch–Delta harboring CHO cells) were patterned in coculture for signal propagation studies. (d) Top panel: Annulus fibronectin pattern (red) and surrounding Neutravidin pattern (green). Bottom panel: HUVECs labeled with CellTracker Red were seeded on the fibronectin pattern; once the fibronectin annulus was completely seeded, biotinylated fibronectin was added and HUVECs labeled with CellTracker Green were seeded on the “switched” Neutravidin regions. (e) Top panel: Sinusoidal wave patterns of fibronectin (red) and Neutravidin (green). Bottom panel: HUVECs seeded as in part d. All scale bars, 100 μm.

Figure 5

Patterning interfacial juxtacrine signaling. Tetracycline-inducible Delta expressing sender cells were patterned on a vertical 10 μm wide fibronectin line, followed by Notch receptor cells with yellow fluorescent protein (YFP) reporters of Notch activity on the horizontal 10 μm wide Neutravidin lines. (a) Before addition of tetracycline, no cells express Delta and therefore no cells harbor Notch activity, as evidenced by baseline YFP fluorescence. (b) Delta is induced in sender cells upon addition of tetracycline, which then activates Notch signaling in neighboring receiver cells, visualized as YFP expression localized to the intersection of the vertical and horizontal lines approximately 24 h after addition of tetracycline. (c) Average YFP pixel intensity profiles (taken from the entire images in parts a and b) demonstrate peak Notch activation at the interface between sender and receiver cells. All scale bars, 75 μm.