In order to better understand the brain and brain diseases, in vitro human brain models need to include not only a chemically and physically relevant microenvironment, but also structural network complexity. This complexity reflects the hierarchical architecture in brain tissue. Here, a method has been developed that adds complexity to a 3D cell culture by means of nanogrooved substrates. SH-SY5Y cells were grown on these nanogrooved substrates and covered with Matrigel, a hydrogel. To quantitatively analyze network behavior in 2D neuronal cell cultures, we previously developed an automated image-based screening method. We first investigated if this method was applicable to 3D primary rat brain cortical (CTX) cell cultures. Since the method was successfully applied to these pilot data, a proof of principle in a reductionist human brain cell model was attempted, using the SH-SY5Y cell line. The results showed that these cells also create an aligned network in the 3D microenvironment by maintaining a certain degree of guidance by the nanogrooved topography in the z-direction. These results indicate that nanogrooves enhance the structural complexity of 3D neuronal cell cultures for both CTX and human SH-SY5Y cultures, providing a basis for further development of an easy access brain-on-chip model.

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Brain-on-chip (BOC) technology such as nanogrooves and microtunnel structures can advance in vitro neuronal models by providing a platform with better means to maintain, manipulate and analyze neuronal cell cultures. Specifically, nanogrooves have been shown to influence neuronal differentiation, notably the neurite length and neurite direction. Here, we have drawn new results from our experiments using both 2D and 3D neuronal cell culture implementing both flat and nanogrooved substrates. These are used to show a comparison between the number of cells and neurite length as a first indicator for valuable insights into baseline values and expectations that can be generated from these experiments toward design optimization and predictive value of the technology in our BOC toolbox. Also, as a new step toward neuronal cell models with multiple compartmentalized neuronal cell type regions, we fabricated microtunnel devices bonded to both flat and nanogrooved substrates to assess their compatibility with neuronal cell culture. Our results show that with the current experimental protocols using SH-SY5Y cells, we can expect 200 – 400 cells with a total neurite length of approximately 4,000–5,000 μm per 1 mm 2 within our BOC devices, with a lower total neurite length for 3D neuronal cell cultures on flat substrates only. There is a statistically significant difference in total neurite length between 2D cell culture on nanogrooved substrates versus 3D cell culture on flat substrates. As extension of our current BOC toolbox for which these indicative parameters would be used, the microtunnel devices show that culture of SH-SY5Y was feasible, though a limited number of neurites extended into microtunnels away from the cell bodies, regardless of using nanogrooved or flat substrates. This shows that the novel combination of microtunnel devices with nanogrooves can be implemented toward neuronal cell cultures, with future improvements to be performed to ensure neurites extend beyond the confines of the wells between the microtunnels. Overall, these results will aid toward creating more robust BOC platforms with improved predictive value. In turn, this can be used to better understand the brain and brain diseases.

A new generation of in vitro human brain models is vital to surpass the limitations of current cell culture platforms and animal cell lines in studying brain function and diseases. Brain-on-chip technology can generate well-defined and reproducible platforms to control the cellular microenvironment for in vivo-like, organized brain cell cultures. Previously, the authors investigated differentiation and network organization of the neuroblastoma SH-SY5Y cell line on nanogrooved substrates, showing that nanogroove guidance of neuronal outgrowths is dependent on nanogroove dimensions. Further, increased orientation of neurites was positively correlated to the differentiation of SH-SY5Y cells. However, as mimicking brain structure alone is insufficient, here, the function of the neuronal cell network as dependent on surface topography and material stiffness is investigated. A generalized replication protocol was developed to create similar nanogrooved patterns in cell culture substrates from different materials, specifically polydimethylsiloxane (PDMS) and Ostemer. Experiments using calcium imaging, where calcium fluxes across membranes are visualized as an indication of action potentials in neuronal cells, were performed with differentiated SH-SY5Y cells and human induced pluripotent stem cell-derived neuronal cells (hiPSCNs) on flat versus nanogrooved substrates to study the network function. Calcium live-imaging was performed and results for experiments with SH-SY5Y cells and hiPSCNs showed that nanogrooved PDMS substrates trended toward increased cellular activity and neuronal cell network connectivity. For future investigation of compatible substrate materials in combination with the effect of material stiffness on the cells, nanogrooved Ostemer substrates were demonstrated to faithfully replicate for use in neuronal cell cultures using nanogrooved substrates. First experiments into the neuronal cell function using stem cells described here aid toward elucidating the effect of nanotopographical and mechanical properties and their benefits toward advancing in vitro neuronal cell models both in form and function. Overall, the results indicate, in conjunction with the previous findings on neuronal outgrowth guidance, that anisotropy as introduced by nanogrooved substrates can have a controllable and potentially beneficial influence on neuronal cell cultures.

Research on neuronal differentiation and neuronal network guidance induced through nanotopographical cues generates large datasets, and therefore the analysis of such data can be aided by automatable, unbiased image screening tools. To link such tools, we present an image-based screening method to evaluate the influence of nanogroove pattern dimensions on neuronal differentiation. This new method consists of combining neuronal feature detection software, here HCA-Vision, and a Frangi vesselness algorithm to calculate neurite alignment values and quantify morphological aspects of neurons, which are measured via neurite length, neuronal polarity, and neurite branching, for differentiated SH-SY5Y cells cultured on nanogrooved polydimethylsiloxane (PDMS) patterns in the 200–2000 nm range. The applicability of this method is confirmed by our results, which find that the level of alignment is dependent on nanogroove dimensions. Furthermore, the screening method reveals that differentiation and alignment are correlated. In particular, patterns with groove widths >200 nm and with a low ridge width to pattern period ratio have a quantifiable influence on alignment, neurite length, and polarity. In summary, the novel combination of software that forms a base for this statistical analysis method demonstrates good potential for evaluating tissue microarchitecture, which depends on subtle design variation in substrate topography. Using the screening method, we obtained automated and sensitive quantified readouts from large datasets.