Neural cell alignment by patterning gradients of the extracellular matrix protein laminin

Abstract

Anisotropic orientation and accurate positioning of neural cells is achieved by patterning stripes of the extracellular matrix protein laminin on the surface of polystyrene tissue culture dishes by micromoulding in capillaries (MIMICs). Laminin concentration decreases from the entrance of the channels in contact with the reservoir towards the end. Immunofluorescence analysis of laminin shows a decreasing gradient of concentration along the longitudinal direction of the stripes. The explanation is the superposition of diffusion and convection of the solute, the former dominating at length scales near the entrance (characteristic length around 50 μm), the latter further away (length scale in excess of 900 μm). These length scales are independent of the channel width explored from about 15 to 45 μm. Neural cells are randomly seeded and selectively adhere to the pattern, leaving the unpatterned areas depleted even upon 6 days of incubation. Cell alignment was assessed by the orientation of the long axis of the 4',6-diamidino-2-phenylindole-stained nuclei. Samples on patterned the laminin area exhibit a large orientational order parameter. As control, cells on the unpatterned laminin film exhibit no preferential orientation. This implies that the anisotropy of laminin stripes is an effective chemical stimulus for cell recruiting and alignment.

Keywords: laminin; neural cells; patterning; protein gradients; soft lithography.

Figure 1.

Laminin patterning on polystyrene TC dishes. (a) A schematic of the MIMIC patterning technique; (b) layout of the mask used for lithography: white lanes represent the channels at increasing widths (c) optical- (d) immunofluorescence- and (e) scanning probe micrographs with line profile (inset) of the patterned laminin stripes.

Figure 2.

Laminin gradient analysis. (right panel) Velocity fields calculated on the horizontal plane of microchannels with width equal to 18, 25 and 41 μm; (left panel) representative data of laminin fluorescence intensities (y-axis, left) versus stripe length are shown for the same stripes. The black dots outline the decrease in fluorescence intensity. Concentration of adsorbed laminin (y-axis, right), obtained from finite-element calculation (see the electronic supplementary material), is plotted versus stripe length (continuous lines). The concentration gradient is increasing with time during MIMICs deposition.

Figure 3.

Inverse decay lengths of the double exponential fit extracted from the analysis of laminin fluorescence intensity. k₁ describes the slope of gradient at large distances from the reservoir (filled symbol), whereas k₂ regulates the second slope close to the reservoir (open symbol). Values are expressed as mean±s.e.m.

Figure 4.

Optical and fluorescence micrographs of 1321N1 astrocytoma cells after 6 days of incubation under standard cell culture conditions (37°C, 5% CO₂, 90% RH). Nuclei of 1321N1 astrocytoma cells stained with DAPI on laminin-unpatterned (a) and -patterned (b) regions of polystyrene TC dishes; (d) higher magnification images showing details of nuclei organization in parallel lanes onto laminin stripes with increasing widths. In (c,e), optical images of the patterned culture where the whole cell shape can be appreciated.

Figure 5.

Analysis of the orientation angles of 1321N1 cell nuclei. (a) Definition of the orientation angle Θ of nucleus along the laminin stripe direction. Polar plots of angle values for cells on laminin-unpatterned (b) and -patterned (c) regions of TC dishes (representative example for stripe with 25.8 μm nominal width is shown). (d) Percentage population of nuclei with angle values −1° < Θ < 1° (indicating high grade of alignment) versus stripe width, estimated in region closer (white bars) and further from the reservoir (black bars).

Figure 6.

Correlation between order parameter S = < cos2Θ > versus exponential decay length (k₁ and k₂) of the gradients. Values corresponding to the region close to (squares) and far from (triangles) the reservoir. (Online version in colour.)