Three-dimensional (3D) neuronal cell culture models are complex in vitro systems that allow earlier neurite outgrowth and functional network formation than with 2D neuronal cultures [1]. Such 3D neuronal models can allow the study of neurological/neurodegenerative diseases in comparison to the healthy condition. In particular, the third dimension generates a microenvironment closer to the in vivo situation in the brain. Native brain tissue has a low elastic modulus which varies between 30-500 Pa depending on the brain area and the stage of development [2,3]. We and others have demonstrated that neuronal network formation is highest in ultrasoft hydrogels [1,4].

Here, we have used thiolated hyaluronic acid (HA-SH) crosslinked with poly (ethylene glycol) diacrylate. Hyaluronic acid – one major component of the brain’ extracellular matrix – was reinforced by box pore frames composed of poly(ɛ-caprolactone) (PCL) fibres and made via melt electrowriting (MEW). These 9-mm diameter circular MEW reinforcement frames were made with 10-layers of 9.7 ± 0.2 µm diameter fibres spaced 200 µm apart in the x- and y-directions.

Using cell viability as a first readout, we found that neither the addition of Maxgel, proteoglycans, peptides (RGD, YIGSR, IKVAV), nor brain-derived neurotrophic factor or ciliary neurotrophic factor showed any significant positive effect on the growth of cortical neurons in reinforced-HA-SH hydrogels. When adding a fraction (20-25%) of Matrigel, neurons exhibited enhanced viability. To overcome this issue, astrocytes which provide essential components of the brain ECM and produce growth factors that enable neuronal path finding and survival were seeded as a 2D layer below the 3D reinforced hydrogels containing cortical neurons. Interestingly, astrocytes significantly increased neuronal survival but also were detected within the 3D construct in close contact to cortical neurons. Thus, astrocytes represent an essential component in the 3D MEW-frame HA-SH constructs and support neuronal network formation and function. Neuronal network formation was quantified by neurite densities, and synaptic puncta increase and further functionally investigated by action potential firing and Ca²⁺ imaging. Thus, our 3D MEW-frame HA-SH composite with neurons as well as astrocytes represents a suitable model to further study brain diseases by comparing healthy and disease conditions at the structural and functional level.