Epithelial contact guidance on well-defined micro- and nanostructured substrates

Abstract

The human corneal basement membrane has a rich felt-like surface topography with feature dimensions between 20 nm and 200 nm. On the basis of these findings, we designed lithographically defined substrates to investigate whether nanotopography is a relevant stimulus for human corneal epithelial cells. We found that cells elongated and aligned along patterns of grooves and ridges with feature dimensions as small as 70 nm, whereas on smooth substrates, cells were mostly round. The percentage of aligned cells was constant on substrate tomographies with lateral dimensions ranging from the nano- to the micronscale, and increased with groove depth. The presence of serum in the culture medium resulted in a larger percentage of cells aligning along the topographic patterns than when no serum was added to the basal medium. When present, actin microfilaments and focal adhesions were aligned along the substrate topographies. The width of the focal adhesions was determined by the width of the ridges in the underlying substrate. This work documents that biologic length-scale topographic features that model features encountered in the native basement membrane can profoundly affect epithelial cell behavior.

Fig. 1

SEM images of human corneal epithelial cells. (A) Cell cultured on a silicon oxide substrate patterned with 70 nm wide ridges, on a 400 nm pitch. The groove depth was 600 nm. (B) Cell on a smooth silicon oxide substrate.

Fig. 2.

Distribution of orientation angles for cells cultured on substrates patterned with 70 nm wide ridges and 600 nm deep grooves, on a 400 nm pitch (circles), and for cells cultured on smooth silicon oxide substrates (triangles). Each data point corresponds to the percentage of cells that have orientation angles in the 10° angle interval up to the coordinate in the x-axis.

Fig. 3

Average cell elongations (cell length/cell breadth) of cells cultured on substrates patterned with 70 nm wide ridges and 600 nm deep grooves, on a 400 nm pitch (circles) and of cells on smooth substrates (triangles). Average elongation of nonaligned cells on patterned substrates (cells with orientation angles between 10° and 90°) was equivalent to the average elongation of cells cultured on smooth substrates. Cells with orientation angles less than 10° were on average significantly more elongated than nonaligned cells cultured on the patterned substrates or cells cultured on smooth substrates.

Fig. 4

Histogram of cell elongations for cells cultured on substrates patterned with 70 nm wide ridges and 600 nm deep grooves, on a 400 nm pitch and for cells cultured on smooth silicon oxide substrates. On the patterned substrates more than 80% of the cells with elongations higher than 2 were aligned along the substrate patterns. There was no correlation between cell elongation and the percentage of cells with orientation angles less than 10° on the smooth substrates.

Fig. 5

Cells extended and retracted lamellipodia along the direction of the patterns. (A-D) Time-lapse microscopy of a cell cultured on grooves and ridges on a 400 nm pitch. The direction of the patterns can be clearly seen but, due to the resolution of these images, the observed pitch is not correct. (E-H) Cell on smooth silicon oxide. (A,E) 2.5 hours; (B,F) 9.5 hours; (C,G) 10.5 hours; (D,H) 12 hours after cell seeding.

Fig. 6

Cells on smooth substrates had longer trajectories than cells on substrates with grooves and ridges on a 400 nm pitch. Cell trajectories correspond to the coordinates of the cells' centroids, recorded every 30 minutes from 2 hours until 12 hours after seeding. The units of the axes are micrometers. (A) The centroids of cells on patterned substrates were mostly stationary. (B) On smooth substrates cells had random trajectories.

Fig. 7

Percentage of cells cultured on patterned and smooth substrates that had orientation angles less than 10° (designated aligned cells). On substrates with 600 nm deep grooves, the percentage of aligned cells was constant on patterns with pitches ranging from 400 nm to 2000 nm and decreased on 4000 nm pitch patterns. On 150 nm deep grooves, cell alignment was equivalent on all pattern pitches and was significantly lower than on 600 nm deep grooves, for pitches up to 2000 nm.

Fig. 8

Average projected cell areas of elongated cells (cell elongations higher than 1.3) and round cells (cell elongations lower than 1.3) on substrates patterned with 600 nm deep grooves and pitches between 400 nm and 4000 nm and on smooth substrates (SiOx and TCPS). The culture medium was SHEM with 10% FBS. The average projected cell areas of both elongated and round cells on any of the patterned surfaces were lower than on the smooth surfaces.

Fig. 9

Percentage of aligned cells in the presence or absence of FBS. When FBS was not added to the culture medium the percentage of aligned cells was equivalent on all pattern pitches and was significantly lower than in the presence of serum, for pitches up to 2000 nm.

Fig. 10

Average projected cell areas of elongated cells (cell elongations higher than 1.3) and round cells (cell elongations lower than 1.3) on substrates patterned with 600 nm deep grooves and pitches between 400 nm and 4000 nm and on smooth substrates (SiOx and TCPS). The culture medium was SHEM and no FBS was added. The average projected cell areas were similar on SiOx and on TCPS.

Fig. 11

SEM images of cells cultured on patterns with 400 nm pitch. (A) Cell aligned along nanostructured substrate. (B) Cross-sectional image of cell patterned substrate. (C) Filopodia extend along the top of ridges and bottom of grooves. Lamellipodia protrude into the grooves at the cell edge along the topographic patterns (bottom left) but bridge the grooves at the leading edge of the cell.

Fig. 12

SEM images of cells cultured on patterns with 4000 nm pitch. (A) Cell aligned along microstructured substrate. (B) At the cell edges perpendicular to the patterns, lamellipodia were able to adhere to the floor of the grooves on 2100 nm wide grooves.

Fig. 13

Cells stained for actin (red), vinculin (green) and the nucleus (blue) as described in Materials and Methods. (A) Cell on a substrate with 600 nm deep grooves and 70 nm wide ridges on a 400 nm pitch. (B) Cell on a substrate with 600 nm deep grooves and 1900 nm ridges on a 4000 nm pitch. A reflection image of the substrates is included in the figure insets. (C) Cell on a smooth silicon oxide substrate.

Fig. 14

Average focal adhesion widths for cells cultured on smooth or patterned silicon oxide substrates. The ridge width for each of the patterned substrates is indicated (open squares).