Nanogroove-enhanced hydrogel scaffolds for 3D neuronal cell culture: an easy access brain-on-chip model
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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Exploiting nanogroove-induced cell culture anisotropy to advance in vitro brain models
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.
Retinoic acid accelerates the specification of enteric neural progenitors from in vitro-derived neural crest
The enteric nervous system (ENS) is derived primarily from the vagal neural crest, a migratory multipotent cell population emerging from the dorsal neural tube between somites 1-7. Defects in the development and function of the ENS give rise to a range of disorders, termed enteric neuropathies and include conditions such as Hirschsprung’s disease. Little is known about the signalling that specifies early ENS progenitors. This has, thus far, limited progress in the generation of enteric neurons from human Pluripotent Stem Cells (hPSCs) that could provide a useful tool for disease modelling and regenerative medicine. We describe the efficient and accelerated generation of ENS progenitors from hPSCs, revealing that retinoic acid is critical for the acquisition of both vagal axial identity and early ENS progenitor specification. These ENS progenitors generate enteric neurons in vitro and following in vivo transplantation, achieving long-term colonisation of the ENS in adult mice. Thus, hPSC-derived ENS progenitors may provide the basis for cell therapy for defects in the ENS.