In order to get a better understanding of amoeba-substrate interactions in the processes of cellular adhesion and directional movement, we engineered glass surfaces with defined local adhesion characteristics at a micrometric scale. Amoeba (Dictyostelium dicoideum) is capable to adhere to various surfaces independently of the presence of extracellular matrix proteins. This paper describes the strategy used to create selective adhesion motifs using an appropriate surface chemistry and shows the first results of locally confined amoeba adhesion. The approach is based on the natural ability of Dictyostelium to adhere to various types of surfaces (hydrophilic and hydrophobic) and on its inability to spread on inert surfaces, such as the block copolymer of polyethylene glycol and polypropylene oxide, named Pluronic. We screened diverse alkylsilanes, such as methoxy, chloro and fluoro silanes for their capacity to anchor Pluronic efficiently on a glass surface. Our results demonstrate that hexylmethyldichlorosilane (HMDCS) was the most appropriate silane for the deposition of Pluronic. A complex dependence between the physicochemistry of the silanes and the polyethylene glycol block copolymer deposition was observed. Using this method, we succeed in scaling down the micro-fabrication of pluronic-based adhesion motifs to the amoeba
Deep Dive into Microscale adhesion patterns for the precise localization of amoeba.
In order to get a better understanding of amoeba-substrate interactions in the processes of cellular adhesion and directional movement, we engineered glass surfaces with defined local adhesion characteristics at a micrometric scale. Amoeba (Dictyostelium dicoideum) is capable to adhere to various surfaces independently of the presence of extracellular matrix proteins. This paper describes the strategy used to create selective adhesion motifs using an appropriate surface chemistry and shows the first results of locally confined amoeba adhesion. The approach is based on the natural ability of Dictyostelium to adhere to various types of surfaces (hydrophilic and hydrophobic) and on its inability to spread on inert surfaces, such as the block copolymer of polyethylene glycol and polypropylene oxide, named Pluronic. We screened diverse alkylsilanes, such as methoxy, chloro and fluoro silanes for their capacity to anchor Pluronic efficiently on a glass surface. Our results demonstrate that h
Introduction Cell-substrate dependant polarization and directional movement are crucial for many physiological processes such as embryonic development, wound healing and functional immune [1] and neural [2] systems.
The ability to engineer adequate chemical surfaces for specific cell-surface interactions like spatial control over cell polarization and directional movement has tremendous potential. It opens the way to controlled and predictable host biomaterial interactions that can find an application in tissue engineering and in the development of new biomedical implant materials.
In order to study polarization and cell movement on specific adhesive patterns, we selected the commonly used eukaryotic model cell Dictyostelium discoideum, a social amoeba. It is amenable to routine genetic techniques (directed and random mutagenesis, deletion and over-expression of genes, complementation) and its genome has been fully sequenced [3]. Moreover many of the Dictyostelium genes show a high degree of sequence similarity to genes in vertebrate species and the molecular machineries driving adhesion and cell movement are broadly conserved. These characteristics together with the ease of its culture conditions make Dictyostelium an organism of choice for the study of cell adhesion and movement (for a review, see [4]). In order to control surface adhesion spatially we made use of Dictyostelium’s natural capacity to adhere to almost all types of surfaces and its inability to adhere to special, so-called inert surfaces, such as the block copolymer of polyethylene glycol and polypropylene oxide, named Pluronic [5].
The paper describes the surface chemistry used to create selective adhesion patterns. Different surface characterization results used to select these surface treatments are discussed.
Experimental details 2.1. Pattern microfabrication and surface functionalization.
Cleaned glass cover slips (25 × 60 mm) were washed with ethanol in an ultrasonic water bath during 5 min. The slides were treated with oxygen plasma (Fig. 1a), 100 sccm O 2 , in a LAM 9400 SE reactor at 50mT for 1 min in order to render the glass very hydrophilic (water contact angle in the range of 10° to 20°). The surfaces were covered with positive UV-sensitive resist, S1813 (Fig. 1b) (Shipley Company Inc, USA). The resist was spin-coated and cured according to the manufacturer’s protocol to form a uniform UV-sensitive film of 6.5µm thickness. Standard contact photolithography (Fig. 1c) using 4-inch chromium masks was then used (UV light: Karl Süss aligner MJB4, SUSS MicroTec, Saint-Jeoire, France at 365 nm and 100 mJ/cm 2 ) and the irradiated pattern was revealed with MF-319 developer (Fig. 1d).
An efficient deposition of Pluronic F127 requires surfaces with water contact angles of about 80° on which Pluronic F127 forms an inert monolayer [5,6]. On the opposite, a hydrophilic surface induces formation of surface aggregates [7] that are weakly attached to the substrate surface. Thus on hydrohpilic surfaces Pluronic F127 is little or not at all adsorbed [6]. Consequently, after the removal of the unpolymerized resist, a hydrophobic layer (Fig. 1e) was added by vapor deposition using diverse silanes (as listed bellow) or CH 4 or C 4 F 8 plasma treatment. This resulted in the formation of either hydrophobic surfaces (water contact angles in the range of 90°-110°) or slightly less hydrophobic surfaces (water contact angles in the range of 75°-90°) (Fig. 1f), depending on the type of silane used. In the end, the polymerized resist was dissolved with acetone in an ultrasonic water bath during 20 min (Fig. 1g), which revealed the hydrophilic pattern under the resist protection (water contact angle in the range of 20° to 30°). The glass slides obtained were treated for 30 min with the anti-adhesive, nonionic copolymer surfactant pluronic F127 (Sigma-Aldrich, 1mg/ml in water) (Fig. 1i).
To obtain highly hydrophobic glass surfaces required for Pluronic F127 deposition, we treated the glass slides in a covered glass chamber for 3 min at 100°C with diverse silanes supplied by ABCR (Karlsruhe, Germany) as follows: MTMS:Methyltrimethoxysilane; DMCS:Dimethylchlorsilane; HMDS :Hexylmethyldichlorsilane; OTCS :Octadecyltrichlorosilane PFES : Perfluorodecyltriethoxysilane PFCS : Perfluorodecyltrichlorooxysilane TFHS : Tridecafluorotetrahydrooctyldimethylchlorosilane; 2MPEPS: 2methoxy(propyethoxylenoxy)propyltrimethoxysilane.
Positive resist
OH OH OH OH OH OH OH
OH OH OH OH OH OH OH
Positive resist
Similarly, deposition of a commercial fluoro silane product, OPTOOL DSX TM from Daikin was performed according to the manufacturer’s protocol.
Alternatively, we treated the glass slides with CH 4 plasma (30 sccm CH 4 with 100 sccm Ar, in a LAM 9400 SE reactor at 10mT for 5sec) or C 4 F 8 plasma (30 sccm C 4 F 8 with 100 sccm Ar, in a LAM 9400 SE reactor at 10mT for 5 sec). It has been reported that both plasma treatments result in the formation of methyl and tef
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