Estimation of the Angles Migrating Cells Turn on Three-Dimensional Micro-Patterned Scaffolds by Live Cell Imaging with an Inverted Microscope

Abstract

To determine how the three-dimensional (3D) shapes of scaffolds influence cell migration, 3D micro-patterned scaffolds with various shapes were fabricated on a silicon substrate (725 µm thick, 10 mm×10 mm quadrate) by using photolithography. We imaged living cells on a silicon substrate over 72 h using a novel simple method. NIH-3T3 cells were adhered to the silicon substrate, which was then placed face-down or face-up into culture medium in a 35 mm (12ϕ) glass-bottomed dish. In this method, there is a sufficient gap (1.3 mm) between the downward-facing cells and the bottom of the plate for the culture medium to diffuse over the cells. Cell growth over 72 h was similar in both conditions. NIH-3T3 cells were adhered to three kinds of 3D micro-patterned scaffolds, placed face-down into culture medium in glass-bottomed dishes, and cell migration and the scaffolds were observed over 72 h. The three scaffolds differed only in terms of the unit shape of the repetitive pattern, namely a scale structure with equilateral triangular pores, a check structure with regular tetragonal pores, or a stripe structure with rectangular grooves. These scaffolds had a constant pore ratio (50%), pore depth (22 µm), and subcellular pattern size. The angle at which cells turned correlated with the unit shape of the scaffold: the interior turning angles were multiples of 60 • on a scale structure with equilateral triangular pores, multiples of 45 • on a check structure with regular tetragonal pores, and close to 0 • or 180 • on a stripe structure with rectangular grooves. Therefore, the angle that cells turn is influenced by the unit shape of the 3D patterned scaffold on which they are cultured. Furthermore, when the angles at which the migrating cells turned were investigated in detail, it was found that a cell turns in one of two directions that correlate with the unit shape of the scaffold; one corresponding to the edge of the pattern, and the other corresponding to the upper surface of the pattern. These differences in the angles that migrating cells turned correlated with differences in the angles they extended protrusions. The angles of cell protrusions markedly differed between the three different scaffolds, which partly underlies why migrating cells turned at different angles. In summary, the unit shape of the micro-patterned scaffold affects the angle at which cells extend, which in turn affects the angle at which migrating cells turn.

Modulation of surface stiffness and cell patterning on polymer films using micropatterns

“…Cell culture scaffolds consisted of a flat, elastic PCL layer (see below) adhered to a micropattern. The micropatterns were fabricated on a square silicon substrate (725 μm thick, 10 × 10 mm) by photolithography . Deep etching of the silicon substrate was performed using the Bosch process for deep reactive ion etching.…”

“…The micropatterns were fabricated on a square silicon substrate (725 lm thick, 10 3 10 mm) by photolithography. 39,40 Deep etching of the silicon substrate was performed using the Bosch process for deep reactive ion etching. Patterns were generated in nine regions (2 3 2 mm) of the substrate, and each patterned area was 500 lm from the adjacent patterned areas.…”

A 3D Microfabricated Scaffold System for Unidirectional Cell Migration

“…To develop a 3D shape that could induce unidirectional cell migration, we focused on the tendency of cells to adhere to 3D sharp edges, [12][13][14] in which cells extend protrusions along the edges of micropatterns. [13,15,16] Cells are known to migrate in the direction of protrusion extension. [17,18] We therefore hypothesized that developing a sharp edge configuration in which the direction of cell protrusions is regulated would be able to direct cell migration.…”

“…To induce unidirectional cell migration using a micropattern 3D shape, we predicted that an edge structure in which cells put forth protrusions and an anisotropic periodic structure with a size comparable to cell size or subcellular size would be important. [13][14][15][16] We also predicted that a device that restricts the direction of cell migration to one dimension would also be important. To restrict the direction of cell migration to one dimension, we decided to employ a stripe-like pattern, which was previously reported to restrict the direction of cell migration to one dimension.…”

“…To restrict the direction of cell migration to one dimension, we decided to employ a stripe-like pattern, which was previously reported to restrict the direction of cell migration to one dimension. [15] First, we combined a scale-like pattern [15,16] ( Figure 1A1), corresponding to a micropattern with an anisotropic periodic structure, with a stripe-like pattern [15] ( Figure 1A2) into a unique and uniform pattern ( Figure 1A3) to restrict the direction of cell migration to one dimension. Rows of the unique pattern on a triangular prism were arranged in a stripe-like pattern.…”