3D Microperiodic Hydrogel Scaffolds for Robust Neuronal Cultures

Abstract

Three-dimensional (3D) microperiodic scaffolds of poly(2-hydroxyethyl methacrylate) (pHEMA) have been fabricated by direct-write assembly of a photopolymerizable hydrogel ink. The ink is initially composed of physically entangled pHEMA chains dissolved in a solution of HEMA monomer, comonomer, photoinitiator and water. Upon printing 3D scaffolds of varying architecture, the ink filaments are exposed to UV light, where they are transformed into an interpenetrating hydrogel network of chemically cross-linked and physically entangled pHEMA chains. These 3D microperiodic scaffolds are rendered growth compliant for primary rat hippocampal neurons by absorption of polylysine. Neuronal cells thrive on these scaffolds, forming differentiated, intricately branched networks. Confocal laser scanning microscopy reveals that both cell distribution and extent of neuronal process alignment depend upon scaffold architecture. This work provides an important step forward in the creation of suitable platforms for in vitro study of sensitive cell types.

Figure 1

Structure and rheological behavior of pHEMA ink. (a) Schematic illustration of pHEMA ink before and after photopolymerization; blue chains represent physically entangled, high molecular weight species and red chains represent chemically cross-linked species (b) Ink viscosity as a function of shear rate; solid black line denotes the shear thinning exponent. (c) Ink shear elastic (G′) and viscous (G″) moduli measured in oscillatory mode at 1 Hz.

Figure 2

SEM micrographs of 3D pHEMA scaffolds of varying architecture, with pitch of a) 30 μm, b) 40 μm, c) 60 μm, and d) 80 μm. All scale bars are 20 μm.

Figure 3

Confocal images (x-y scans, tiled) of primary rat hippocampal cells distributed within scaffolds of varying pitch: (a) 30 μm, (b) 40 μm, (c) 60 μm, and (d) 80 μm. A primary monoclonal antibody for actin is used to label the processes (green), while TO-PRO3 was used to label nuclei (red). Side view reconstructions schematically denote the positions (in x-z plane) of the neuronal somata, while their relative size indicates their position along the y-axis. Scale bar, 40 μm.

Figure 4

Cell distribution as a function of (a) lateral and (b) vertical position within 3D scaffolds of varying pitch.

Figure 5

Confocal images of representative neuronal cells on the scaffolds (top row), reconstructed views (middle row) and reconstructed view of cells only (bottom row). (a) Pyramidal soma morphology on scaffold (60 μm pitch). (b) Neuronal process wrapping around cylindrical feature within scaffold (60 μm pitch). (c) Soma supported by neuronal processes on scaffold (40 μm pitch). (d) Process contact guidance on a scaffold (30 μm pitch). In the confocal images, the scaffold and cell nuclei are stained red and the processes are green. In the reconstructions, nuclei are colored blue, while actin is colored green. Scale bar, 20 μm.

Figure 6

Planar representations of 3D fast Fourier transform (FFT) calculated for (a) scaffold with 40 μm pitch and (b) corresponding neuronal processes on this scaffold. Inset in (a) is an SEM image that indicates scaffold orientation. Normalized intensity as a function of angle for (c) scaffolds of varying pitch and (d) neuronal processes calculated from the FFTs. (e) Extent of process alignment as a function of scaffold pitch. (f) Line scans of intensity measured across the primary angle of the neuronal process alignment in the FFTs (denoted by arrows in (b)). Scale bar, 20 μm.