Exploiting nanogroove-induced cell culture anisotropy to advance in vitro brain models

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.

Nanogrooves for 2D and 3D Microenvironments of SH-SY5Y Cultures in Brain-on-Chip Technology

“…With this in mind, we do expect that extending the indicative results for neuronal cell cultures in 2D and 3D on nanogrooved and flat substrates should also be extended toward human induced pluripotent stem cell-derived neurons. We have previously performed experiments with such cells on nanogrooves ( Bastiaens et al, 2019a ), however, this was done in the context of their electrophysiological assessment using calcium imaging, not on morphological features such as neurite length. With the addition of clear expectations for valuable parameters that indicate neuronal differentiation and the potential to implement human induced pluripotent stem cells for more realistic cultures, such a platform lends itself to preclinical drug discovery where both healthy and patient-derived cells can be cultured and analyzed within the BOC platform.…”

“…PDMS was made at a ratio of 10:1 elastomer to curing agent. Specifically, for the results detailed in this work, nanogrooved PDMS substrates with a pattern period of 1,000 nm and a ridge width of 230 nm were used in neuronal cell culture as these patterns had shown the largest influence on neurite alignment ( Bastiaens et al, 2019a ). For 2D neuronal cell culture, the nanogrooved PDMS substrates including flat PDMS surface areas as control were placed in Petri dishes.…”

Analyzing Developing Brain-On-Chip Cultures with the CALIMA Calcium Imaging Tool

“…Furthermore, it is advisable to choose window length K, such that it can contain at least one full calcium spike. The rise times and decay constants of the calcium spikes depend among others on the fluorescent dye [24] and last milliseconds to min [24,25], placing window size K in the range of tens (footage acquisition rate~1 Hz [4,5,25,26]) to hundreds of frames (acquisition rate~10 Hz [27]). Parameter q should be chosen sufficiently high such that multiple samples are used to estimate F low , but well below 50 to avoid basing F 0 on trace sections that contain calcium spikes.…”

“…Additionally, published data from human induced pluripotent stem cell-derived neuronal cells (hiPSCNs) on nanogrooved polydimethylsiloxane (NG-PDMS) was used to demonstrate the functionality of CALIMA 2.0 in a BoC environment [4] (dataset 2). In brief, nanogrooved substrates, obtained via lithography with a 1000 nm pattern periodicity and 230 nm ridge [5], were used to study guidance of neuronal outgrowths in hiPSCNs.…”

“…Another recent trend in the development of BoC cultures is its influence on activities in the cultures by integrated nano-and microfabricated physical features that, either directly affect cell differentiation processes as ECM biomimicking factors, e.g., nanogrooves [4], and by time-responsive control mechanisms to evoke a response of the cultured network [5].…”