Cell interaction study method using novel 3D silica nanoneedle gradient arrays

Abstract

Understanding cellular interactions with culture substrate features is important to advance cell biology and regenerative medicine. When surface topographical features are considerably larger in vertical dimension and are spaced at least one cell dimension apart, the features act as 3D physical barriers that can guide cell adhesion, thereby altering cell behavior. In the present study, we investigated competitive interactions of cells with neighboring cells and matrix using a novel nanoneedle gradient array. A gradient array of nanoholes was patterned at the surface of fused silica by single-pulse femtosecond laser machining. A negative replica of the pattern was extracted by nanoimprinting with a thin film of polymer. Silica was deposited on top of the polymer replica to form silica nanoneedles. NIH 3T3 fibroblasts were cultured on silica nanoneedles and their behavior was studied and compared with those cultured on a flat silica surface. The presence of silica nanoneedles was found to enhance the adhesion of fibroblasts while maintaining cell viability. The anisotropy in the arrangement of silica nanoneedles was found to affect the morphology and spreading of fibroblasts. Additionally, variations in nanoneedle spacing regulated cell-matrix and cell-cell interactions, effectively preventing cell aggregation in areas of tightly-packed nanoneedles. This proof-of-concept study provides a reproducible means for controlling competitive cell adhesion events and offers a novel system whose properties can be manipulated to intimately control cell behavior.

Figure 1. Pattern prepared on fused silica substrate by single-pulse femtosecond laser machining

(A) A schematic of the pattern displaying the 2D gradient in nanohole spacing (nanohole density reduced for visual clarity). The pattern is a 2×2 matrix of four quadrants, each formed by increasing the spacing between successive nanoholes by 1 μm starting from 10 μm in the densest location (edge) to 50 μm in the sparsest location (middle of the quadrant) and then decreasing from 50 μm to 10 μm at 1 μm decrements in both x and y directions. A quadrant is a matrix of 84×84 nanoholes, and a 2×2 matrix of these quadrants forms the pattern with 168×168 nanoholes. Each black dot in the schematic represents a nanohole made by focusing a single laser pulse. (B) An SEM image of the nanoholes from one edge of the pattern.

Figure 2. Silica nanoneedle fabrication

(A) Schematic of the fabrication of silica nanoneedles starting from cellulose acetate (CA) replication of the laser patterned fused silica template (steps I and II). The replica with CA nanoneedles is then peeled off from the template and glued to a 170 μm glass coverslip with uncured PDMS, and cured at room temperature for 24 hours (steps III and IV). The glass coverslip with CA nanoneedles is then affixed to an aluminum SEM peg (step V) and subjected to silica deposition to form silica nanoneedles. The glass coverslip with silica nanoneedles is then detached from the SEM peg by selectively dissolving the sticky tab adhesive in toluene. (B) SEM image of silica pattern in a specific location where nanoneedles are densely spaced in one direction and sparsely spaced in the orthogonal direction. (C) SEM images of silica nanoneedles (taken at 45° stage tilt) from a section of the 2D gradient pattern at one edge illustrating the x and y increment of 1 μm every successive nanohole.

Figure 3. SiO₂ nanoneedles promote cell attachment and spreading

(A) Significantly more cells attach to nanoneedle-containing surfaces and (B) cell viability is unaffected. (C–D) Cell spreading (area, perimeter) are increased on nanoneedle-containing substrates.

Figure 4. Cell adhesion is influenced by the presence and spacing of SiO₂ nanoneedles

(A–B) Flat surfaces promote the formation of large, unorganized cell aggregates. (C–D) Cells interact directly with the nanoneedles and only small cell aggregates, if any, are able to form. (E) Parallel rows of tightly spaced nanoneedles sequester cells between them. (F) Few, if any, cells are able to attach in areas of densely packed nanoneedles, but attachment improves as spacing becomes sparser. Nanoneedle locations are indicated by X’s in each image. A: calcein, green; B–C: αSMA, green; Hoechst, blue; D–F: actin, red; Hoechst, blue.