3D-printed nanostructures for studying neuronal networks

Researchers at Delft University of Technology have developed a 3D-printed model that enables neuronal growth in a brain-like environment. Using tiny nanopillars, the model mimics the soft extracellular matrix of the brain. This system provides new insights into how neurons form networks and could contribute to research into neurological diseases such as Alzheimer’s or Parkinson’s in the future.

Neurons react to the mechanical and geometric properties of their environment. Conventional cell culture dishes are flat and rigid, in contrast to the soft and fibrous structure of the brain. To better replicate this environment, the team led by scientist Angelo Accardo developed a special nanopillar arrangement using a high-precision 3D printing technique called two-photon polymerization. The pillars, a thousand times thinner than a human hair, are arranged in such a way that they simulate mechanical properties that neurons find in their natural environment.

“This tricks the neurons into “thinking” that they are in a soft, brain-like environment, even though the nanopillars’ material itself is stiff. While bending under the crawling of neurons, the nanopillars not only simulate the softness of brain tissue but also provide a 3D nanometric structure that neurons can grab onto, much like the extra-cellular matrix nano-fibers in real brain tissue,” says Accardo. This influences how the neurons grow and connect with each other.

By varying the height and width of the pillars, the researchers can control the shear modulus of the material. The structure tricks neurons into thinking they are in a soft environment, even though the material itself is stiff. This influences the growth and connection of the neurons. Experiments with different cell types showed that neurons grew in a more targeted and organized manner on this 3D-printed matrix than on conventional 2D substrates.

Accardo: “These hand-like structures guide the tips of growing neurons as they search for new connections. On flat surfaces, the growth cones spread out and remain relatively flat. But on the nanopillar arrays, the growth cones sent out long, finger-like projections, exploring their surroundings in all directions — not just along a flat plane but also in the 3D space, resembling what happens in a real brain environment.”

A study published in the journal Advanced Functional Materials also describes that the system promotes neuronal maturation processes. Neuronal precursor cells that grew on the nanopillars showed increased expression of maturation markers. This indicates that the environment influences not only the growth but also the differentiation of the cells.

“The problem is that gel matrices, like collagen or Matrigel, typically suffer from batch-to-batch variability and do not feature rationally designed geometric features. The nanopillar arrays model offers the best of both worlds: it behaves like a soft environment with nanometric features, and holds extremely high reproducibility thanks to the resolution of two-photon polymerization,” explains Accardo.

The model could help to better understand differences between healthy and diseased neuronal networks. While gel matrices were previously considered the standard for soft cell cultures, the printed nanostructures offer greater reproducibility and more precise geometric properties. The project was funded by interdisciplinary collaboration within the Faculties of Mechanical Engineering and Applied Physics at TU Delft and the ErasmusMC.