High-throughput screening of microscale pitted substrate topographies for enhanced nonviral transfection efficiency in primary human fibroblasts

Abstract

Optimization of nonviral gene delivery typically focuses on the design of particulate carriers that are endowed with desirable membrane targeting, internalization, and endosomal escape properties. Topographical control of cell transfectability, however, remains a largely unexplored parameter. Emerging literature has highlighted the influence of cell-topography interactions on modulation of many cell phenotypes, including protein expression and cytoskeletal behaviors implicated in endocytosis. Using high-throughput screening of primary human dermal fibroblasts cultured on a combinatorial library of microscale topographies, we have demonstrated an improvement in nonviral transfection efficiency for cells cultured on dense micropit patterns compared to smooth substrates, as verified with flow cytometry. A 25% increase in GFP(+) cells was observed independent of proliferation rate, accompanied by SEM and confocal microscopy characterization to help explain the phenomenon qualitatively. This finding encourages researchers to investigate substrate topography as a new design consideration for the optimization of nonviral transfection systems.

Figure 1

Topographical library details: 10 pit morphologies (A–J) were replicated with 16 different combinations of size (X, 1–6 μm) and spacing (Y, 1–6 μm), giving 160 unique patterned PDMS substrates for cell growth, each with a uniform pit depth of 2.4 μm. Smooth regions were present in the center of the array (SM), and in the regions between patterns. Pattern K was excluded from all analyses due to poor cell attachment.

Figure 2

(a) Transfection efficiency, (b) spreading, (c) cell number, and (d) proliferation of NHDFs cultured on topographical PDMS libraries normalized to values taken from smooth regions in each array. Data are grouped across pit morphologies A–J by pit diameter (X, left), and pit spacing (Y, right). Letters denote significance between columns and #’s denote significance compared to smooth regions. (a) Small interfeature spacing (Y = 1μm) produced a 25% increase in transfection efficiency compared to smooth regions, while feature size (X) had a less-significant impact. (b) Pits spaced far apart (large Y), and those with small diameters (small X), supported the highest levels of spreading. (c) Feature size (X) had no detectable effect on cell number whereas patterns with large interfeature spacing (Y = 6 μm) contained 25% more cells than those with small interfeature spacing (Y = 1 μm). (d) No significant dependence of proliferation on feature spacing was detected, suggesting increased cell number on features with large interfeature spacing may reflect increased initial attachment.

Figure 3

Representative fluorescence microscopy images of NHDFs transfected on topographical PDMS libraries, with visible GFP transgene (green), actin (phalloidin stain, red), and nuclei (DAPI, blue). Patterns with small interfeature spacing (B(6,1) and I(6,1)) supported higher transfection efficiency, greater alignment, and less spreading than those with large interfeature spacing (B(1,6) and I(1,6)).

Figure 4

Flow cytometry corroborates the results presented in Figure 3a; F(4,1), a pattern with small interfeature spacing (1 μm), supported 25% higher transfection efficiency than smooth PDMS and F(1,4), a substrate with large interfeature spacing (4 μm).

Figure 5

SEM of cells interacting with pitted topography, imaged with 30° tilt. NHDFs cultured on F(1,4) are more spread than those cultured on F(4,1). Regions of the cell membrane were stretched over open pits on both patterns. Cells cultured on F(4,1) were observed to dramatically deform pit sidewalls.

Figure 6

Confocal microscopy revealed NHDFs exploring the bottom of 1 and 4 μm diameter pits. The appearance of “holes” in the cell membrane of cells cultured on pattern F(4,1) is a consequence of the apical and basal cell surfaces bending below the focal plane, whereas cells on F(1,4) only reached the bottom of pits with their basal membrane.