Abstract
In this work, nanogroove dimensions as a design input parameter for neuronal differentiation and neurite outgrowth in brain-on-a-chip (BOC) applications are investigated. Soft lithography in polydimethylsiloxane (PDMS) is used extensively in organ-on-a-chip applications to create environments for in vitro models. As such, here it is used to fabricate cell culture substrates with nanogrooved patterns. Using a newly developed analysis method, the effect of the nanogrooved, biomimetic PDMS substrates is compared with lateral and height variations within the nanometer range as measured by means of atomic force microscopy (AFM). PDMS culture substrates were replicated from a cyclic olefin copolymer template, which was fabricated either directly by thermal nanoimprinting from a jet and flash imprint lithography (J-FIL) resist pattern (process I) on a polished silicon wafer or via an intermediate reactive ion etched all-silicon mold (process II) that was fabricated by using the J-FIL resist pattern as in process I as a mask. To study the interplay between the lateral and height dimensions of nanogrooves on the differentiation process of SH-SY5Y cells, which are a well-established model for neuronal cells that form networks in culture, the authors first characterized the feature sizes of the PDMS substrates received from both processes by AFM. On average, nanogrooved patterns from process I had a 1.8 ± 1.1% decrease in pattern period, a 15.5 ± 12.2% increase in ridge width compared to the designed dimensions, and a height of 95.3 ± 10.6 nm. Nanogrooved patterns for process II had a 1.7 ± 1.7% decrease in pattern period, a 43.1 ± 33.2% increase in ridge width, and a height of 118.8 ± 13.6 nm. Subsequently, they demonstrated that neurite outgrowth alignment was particularly strong if the pattern period was 600 nm or 1000 nm with the additional constraint for these patterns that the ridge width is <0.4 times the pattern period. Increasing pattern height increased the fraction of differentiated cells within the cell culture and increased neurite length, but had no direct impact on outgrowth alignment. This study forms the basis for optimization in the bottom-up engineering of neuronal network architecture, for which specific patterns can be selected to assist in neuronal cell differentiation and direct neurite growth and alignment. Such organized neuronal networks can aid in the design of in vitro assay systems for BOC applications by improving biological response readouts and providing a better understanding of the relationship between form and function of a neuronal network.
Highlights
Considering the number of cell culture repeats needed to set up such a design comparison study, it is important that the quality of nanogrooves per culture substrate used in the experiment is highly reproducible and a suitable nanofabrication method, i.e., soft lithography, is selected for this purpose
We investigated the effect on neuronal properties of nanogrooved PDMS substrates fabricated via two different fabrication processes; a J-FIL resist pattern on a polished silicon wafer and an reactive ion etched (RIE) all-silicon mold, which was fabricated using the same J-FIL resist pattern as a mask and used in parallel to obtain cyclic olefin copolymer (COC) templates through thermal nanoimprinting
Soft lithography of PDMS using the COC templates resulted in PDMS substrates from both processes, where height variations received from the two different fabrication processes were used to our advantage to set up a cell culture experiment and study height as a design input parameter influencing differentiation and neurite outgrowth across 27 patterns of different lateral dimensions
Summary
06J801-2 Bastiaens, Xie, and Luttge: Investigating the interplay of lateral and height dimensions. Previous experiments have shown that nanogrooves with lateral dimensions in the order of 400–600 nm and a height of approximately 118 nm induce a strong alignment of neuronal outgrowths of primary rat cortical cells in culture.. It is our aim to investigate nanogroove dimensions as a design input parameter for neuronal differentiation and neurite outgrowth in BOC applications.. It is our aim to investigate nanogroove dimensions as a design input parameter for neuronal differentiation and neurite outgrowth in BOC applications.8,23 For this purpose, we compared the morphology and differentiation of SH-SY5Y cells on nanogrooves with height variations. The results of the SH-SY5Y cell culture experiments were analyzed through image-based screening of cell morphology and neurite properties for all made patterns. Our study allowed us to investigate nanogroove height variations as a design input parameter in influencing neurite outgrowth for BOC applications. Such organized neuronal networks can aid in the design of in vitro assay systems for BOC applications, for example, by improving the biological response readouts and by providing a better understanding of the relationship between form and function within neuronal networks
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More From: Journal of Vacuum Science & Technology B, Nanotechnology and Microelectronics: Materials, Processing, Measurement, and Phenomena
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