Abstract
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.
Highlights
A new generation of advanced 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 brain diseases
Patterns for the Ostemer substrates had a pattern depth of 92 ± 2.10 nm, a pattern periodicity of 986 ± 1.04 nm, and a ridge width of 270 ± 12.3 nm (n = 4). These results show that nanogrooved patterns at these dimensions can be faithfully replicated for different materials, such as PDMS and Ostemer
This work demonstrates a method for a generalized replication protocol, in which nanogrooves on a nanoresist master are replicated into a primary cyclic olefin copolymer (COC) mold, a secondary PDMS mold, and either into a PDMS or an Ostemer cell culture substrate
Summary
A new generation of advanced 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 brain diseases. These new models can be achieved using brain-on-chip technology, employing microand nanofabrication to create well-defined, controllable, and reproducible platforms with a cellular microenvironment that allows for in vivo-like, spatially organized brain cell culture. We have shown a fabrication process by which nanogrooved patterns could be replicated into Ostemer and potentially various materials other than PDMS, by means of which it becomes possible to decouple the effects of mechanical properties and nanotopography on neuronal cell activity derived from the calcium flux profile. Based on this knowledge, input parameter design can be optimized toward the bottom-up engineering of neuronal cell network form and function. These anisotropic features at the nanoscale can be incorporated, thereby advancing the structure and robustness of brain-on-chip platforms and present us with the formation of in vivo-like brain cell cultures
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