Extension nanostructurées Substrats utilisant la technique de point pour Nanotopographical Modulation of Cell Comportement
Summary
A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.
Abstract
Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.
Introduction
A number of recent findings reveal that substrate nanotopography has pronounced influence on cell behavior, from cell adhesion, spreading and migration, to proliferation and differentiation1-6. For instance, a smaller cell size and lower proliferation rate have been observed in cells cultured on deep nanogratings, even leading to apoptosis although the cell alignment, elongation and migration were enhanced, compared with the flat controls2,7-10. Moreover, nanotopography has been shown to facilitate the differentiation of stem cells into certain lineages such as neuron2,11,12, muscle13, and bone3,4. In addition, because of increasing concerns on the toxicity of engineered nanomaterials14,15, there is a need to incorporate nanotopography into physiologically relevant in vitro models for accurate risk assessment of nanomaterials. To fulfil the biochemical and molecular biology analyses, enough cells are needed to be grown on a large area of nanopatterned substrate. However, conventional nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area.
Self-assembly including colloid lithography16 and polymer demixing17 can readily generate large-area nanostructures at low costs. Because self-assembly relies on interactions between the assembling elements such as colloidal particles and macromolecules, and possible interactions between these elements and substrate, it cannot be a stand-alone method of producing nanostructures with precise spatial positioning and arbitrary shapes18. The accompanied high density of defects is also a drawback. Precise spatial control of nanopatterns can be achieved by employing templated self-assembly, which uses top-down lithographic approaches to provide the topographical and/or chemical template to guide the bottom-up assembly of the assembling elements19-21. Alternative nanofabrication techniques such as step-and-flash lithography22 and a roll-to-roll nanoimprinting lithography23 have been developed but have limited use because of their sophisticated process or the requirement of specialized equipment. Nevertheless, a template or a master mold with defined nanoscale patterns is needed for templated or alternative nanofabrication techniques.
Such templates and master molds are conventionally generated by using focused electron, ion, or photon beam lithography. For instance, electron beam lithography (EBL)24 and focused ion beam lithography25 can generate defined patterns with a sub-5 nm resolution. Two-photon lithography has demonstrated a feature size as small as 30 nm26. Although the focused beam lithography techniques enable generation of well-defined nanoscale structures, the capital investment and the time-consuming, costly process restrict their widespread use in academic research27. Therefore, it is highly desirable to develop enabling yet affordable techniques to produce a large area of nanopatterned surfaces with high fidelity.
We have reported a simple, cost-effective stitch technique for generating a large area of nanopatterned surface from a small well-defined mold28. This protocol provides step-by-step procedure from replication of poly(dimethylsiloxane) (PDMS) molds using an EBL-written pattern, to assembly of multiple PDMS molds into a single large mold, to generation of a master mold on polymeric such as polystyrene (PS) substrates, to production of working substrates. With the expanded nanopatterned substrates, we demonstrated nanotopographical modulation of cell spreading.
Protocol
Representative Results
Discussion
Nous présentons une méthode simple, abordable et polyvalent pour générer une grande surface du substrat nanostructuré. Pour développer fidèlement nanoschémas hautement définis, une grande attention doit être accordée à plusieurs étapes critiques. La première est de couper les PDMS moules multiples. les zones sans motif du moule PDMS doivent être enlevés. En outre, les parois latérales des moules doivent être coupés à la verticale aussi parfaite que possible afin de minimiser les écarts entre les mou…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was partly supported by NSF CBET 1227766, NSF CBET 1511759, and Byars-Tarnay Endowment. We gratefully acknowledge use of the West Virginia University Shared Research Facilities which are supported, in part, by NSF EPS-1003907.
Materials
| JEOL field emission SEM | JEOL | JSM-7600F | EBL |
| E-beam evaporator | Kurt J. Lesker | Model: LAB 18 e-beam evaporator | nickel deposition |
| Trion Minilock III ICP/RIE | Trion technology | Model: Minilock-phantom III | |
| Press machine | PHI Hydraulic Press | Molde: SQ-230H | |
| Spin coater | Laurell Technologies | Modle: WS-400A-6NPP-LITE | |
| CO2 critical dryer | Tousimis | Modle: Autosamdri-815 | |
| Silicon wafer | University Wafer | 1080 | |
| Aluminum plates | McMaster-carr | 9057K123 | |
| Teflon sheets | McMaster-carr | 8711K92 | |
| 100 mm petri dish | FALCON | 353003 | |
| 60 mm petri dish | FALCON | 353004 | |
| Glass coverslip | Fisher Scientific | 12-542-B | |
| Glass slide | Fisher Scientific | 12-550-34 | |
| Disposable weighing boats | Fisher Scientific | 13-735-743 | |
| Glass desiccator | Fisher Scientific | 02-913-360 | |
| Plastic desiccator | Bel-Art Products | F42025-000 | |
| Hotplate | Fisher Scientific | 1110049SH | |
| Tweezer | Ted Pella, inc. | 5726 | |
| Blade | Fisher Scientific | S17302 | |
| Metal blocks | McMaster-carr | ||
| Punch | Brettuns Village Leather Craft Supplies | Arch punch | |
| Poly(methyl methacrylate) | MicroChem | 495 PMMA A4 | |
| PDMS | Dow Corning | Sylgard 184 kit | |
| Polystyrene | Dow Chemical | Styron 685D | |
| 1H,1H,2H,2H-perfluorooctylmethyldichlorosilane | Oakwood Chemical | 7142 | |
| Developer | MicroChem | MIBK/IPA at 1: 3 ratio | |
| Remover | MicroChem | Remover PG | |
| Ethanol | Fisher Scientific | BP2818500 | |
| Toluene | Fisher Scientific | T324-500 | |
| Phosphate buffered saline | Sigma Aldrich | D8537 | |
| Dulbecco’s modified eagle medium | Sigma Aldrich | D5796 | |
| Fetal bovine serum | Atlanta Biologicals | S11550 | |
| Paraformaldehyde | Electron Microsopy Science | 15712-S | |
| Glutaraldehyde | Fisher Chemical | G151-1 | |
| Fibronectin | Corning | 356008 | |
| A549 cells | ATCC | ATCC CCL-185 |
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