Robust method for high-throughput surface patterning of deformable substrates.

We describe a simple and robust method for high-throughput surface patterning of deformable substrates such as silicone rubber films covered with a thin layer of protein and cell antifouling hydrogel (PLL-g-PEG). The irradiation with deep UV (<200 nm) of PLL-g-PEG-coated rubber substrates through a synthetic quartz photomask created micropatterns over a large area of the substrate. Incubation with proteins resulted in stable patterns with high feature resolution. RPE1 cells seeded on fibronectin patterns were constrained for days even after stretching. We also propose the crossbow feature as an interesting example allowing the stretching of normalized stress fibers.

Simple and rapid process for single cell micro-patterning.

We present a simple and environmentally friendly process for cell patterning on glass covered with an ultrathin layer of poly-l-lysine-grafted-polyethylene glycol (PLL-g-PEG) by exposure to deep UV light. The patterned substrates are stable for months in the lab atmosphere before incubation with proteins. Incubation with proteins resulted in well defined patterns, with high feature resolution. RPE-1 cells seeded on fibronectin/fibrinogen-Alexa 488 patterns were constrained for days on the deep UV exposed regions. Finally, large glass plates were patterned with high homogeneity enabling the assembly of micro-patterned microplates in 96-well format.

Optimization of the chromone scaffold through QSAR and docking studies: Identification of potent inhibitors of ABCG2.

The membrane transporter BCRP/ABCG2 has emerged as a privileged biological target for the development of small compounds capable of abolishing multidrug resistance. In this context, the chromone skeleton was found as an excellent scaffold for the design of ABCG2 inhibitors. With the aims of optimizing and developing more potent modulators of the transporter, we herewith propose a multidisciplinary medicinal chemistry approach performed on this promising scaffold. A quantitative structure-activity relationship (QSAR) study on a series of chromone derivatives was first carried out, giving a robust model that was next applied to the design of 13 novel compounds derived from this nucleus. Two of the most active according to the model's prediction, namely compounds 22 (5-((3,5-dibromobenzyl)oxy)-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-4-oxo-4H-chromene-2-carboxamide) and 31 (5-((2,4-dibromobenzyl)oxy)-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-4-oxo-4H-chromene-2-carboxamide), were synthesized and had their biological potency evaluated by experimental assays, confirming their high inhibitory activity against ABCG2 (experimental EC50 below 0.10 µM). A supplementary docking study was then conducted on the newly designed derivatives, proposing possible binding modes of these novel molecules in the putative ligand-binding site of the transporter and explaining why the two aforementioned compounds exerted the best activity according to biological data. Results from this study are recommended as references for further research in hopes of discovering new potent inhibitors of ABCG2.

Comparative study and improvement of current cell micro-patterning techniques.

The original micropatterning technique on gold, although very efficient, is not accessible to most biology labs and is not compatible with their techniques for image acquisition. Other solutions have been developed on silanized glass coverslips. These methods are still hardly accessible to biology labs and do not provide sufficient reproducibility to become incorporated in routine biological protocols. Here, we analyzed cell behavior on micro-patterns produced by various alternative techniques. Distinct cell types displayed different behavior on micropatterns, while some were easily constrained by the patterns others escaped or ripped off the patterned adhesion molecules. We report methods to overcome some of these limitations on glass coverslips and on plastic dishes which are compatible with our experimental biological applications. Finally, we present a new method based on UV crosslinking of adhesion proteins with benzophenone to easily and rapidly produce highly reproducible micropatterns without the use of a microfabricated elastomeric stamp.

Multiprotein Printing by Light-Induced Molecular Adsorption.

Light-induced molecular adsorption of proteins (LIMAP) allows for quantitative sub-micrometer-resolution printing of multiple biomolecules. Surface-bound gradients are patterned within minutes over an entire glass cover-slip. LIMAP is used to perform selective immuno-assays, to dynamically control the adhesion of individual cells, and to achieve hierarchical co-cultures instrumental for tissue engineering.

Engineering the surface properties of microfluidic stickers.

We introduce a simple and effective method to tailor the wetting and adhesion properties of thiolene-based microfluidic devices. This one-step lithographic scheme combines most of the advantages offered by the current methods employed to pattern microchannels: (i) the channel walls can be modified in situ or ex situ, (ii) their wettability can be varied in a continuous manner, (iii) heterogeneous patterning can be easily accomplished, with contact-angle contrasts extending from 0 to 90° for pure water, (iv) the surface modification has proven to be highly stable upon aging and heating. We first characterize the wetting properties of the modified surfaces. We then provide the details of two complementary methods to achieve surface patterning. Finally, we demonstrate the two methods with three examples of applications: the capillary guiding of fluids, the production of double emulsions, and the culture of cells on adhesive micropatterns.

A new micropatterning method of soft substrates reveals that different tumorigenic signals can promote or reduce cell contraction levels.

In tissues, cell microenvironment geometry and mechanics strongly impact on cell physiology. Surface micropatterning allows the control of geometry while deformable substrates of tunable stiffness are well suited for the control of the mechanics. We developed a new method to micropattern extracellular matrix proteins on poly-acrylamide gels in order to simultaneously control cell geometry and mechanics. Microenvironment geometry and mechanics impinge on cell functions by regulating the development of intra-cellular forces. We measured these forces in micropatterned cells. Micropattern geometry was streamlined to orient forces and place cells in comparable conditions. Thereby force measurement method could be simplified and applied to large-scale experiment on chip. We applied this method to mammary epithelial cells with traction force measurements in various conditions to mimic tumoral transformation. We found that, contrary to the current view, all transformation phenotypes were not always associated to an increased level of cell contractility.

External forces control mitotic spindle positioning.

The response of cells to forces is essential for tissue morphogenesis and homeostasis. This response has been extensively investigated in interphase cells, but it remains unclear how forces affect dividing cells. We used a combination of micro-manipulation tools on human dividing cells to address the role of physical parameters of the micro-environment in controlling the cell division axis, a key element of tissue morphogenesis. We found that forces applied on the cell body direct spindle orientation during mitosis. We further show that external constraints induce a polarization of dynamic subcortical actin structures that correlate with spindle movements. We propose that cells divide according to cues provided by their mechanical micro-environment, aligning daughter cells with the external force field.

Protein micropatterns: A direct printing protocol using deep UVs.

The described protocol is a simple method to make protein micropatterns with a micron size resolution. It can be applied to control cell shape and adhesive geometry, and also for any other assay requiring protein patterning. It is based on the use of a photomask with microfeatures to locally irradiate with deep UV light (below 200 nm) an antifouling substrate, making it locally adsorbing for proteins. The entire process can be subdivided into three main parts. The first part describes the design of a photomask. The second part describes the passivation (antifouling treatment) of the substrate, its irradiation, and the binding of proteins. The entire process can be completed in a couple of hours. It requires no expensive equipment and can be performed in any biology lab. The last part describes cell deposition on the micropatterned substrate. We also provide a discussion with pitfalls and alternative techniques adapted to various substrates, including silicone elastomers.

Mechanisms to suppress multipolar divisions in cancer cells with extra centrosomes.

Multiple centrosomes in tumor cells create the potential for multipolar divisions that can lead to aneuploidy and cell death. Nevertheless, many cancer cells successfully divide because of mechanisms that suppress multipolar mitoses. A genome-wide RNAi screen in Drosophila S2 cells and a secondary analysis in cancer cells defined mechanisms that suppress multipolar mitoses. In addition to proteins that organize microtubules at the spindle poles, we identified novel roles for the spindle assembly checkpoint, cortical actin cytoskeleton, and cell adhesion. Using live cell imaging and fibronectin micropatterns, we found that interphase cell shape and adhesion pattern can determine the success of the subsequent mitosis in cells with extra centrosomes. These findings may identify cancer-selective therapeutic targets: HSET, a normally nonessential kinesin motor, was essential for the viability of certain extra centrosome-containing cancer cells. Thus, morphological features of cancer cells can be linked to unique genetic requirements for survival.

Tailoring the porous hierarchy of titanium phosphates.

First hierarchical titanium phosphate (TiPO) materials with multiple porosities of different lengths (meso-macroporous and meso-macro-macroporous) were synthesized by the self-formation process. The further tuning of the porous hierarchy by using the poly(ethylene oxide) surfactant technique was demonstrated. The macroporous structure (50-160 nm in size) of TiPO with mesoporous walls could be self-formed in the absence of any templatable agents, including surfactant molecules. On the basis of spontaneous structurization, the addition of a small quantity of nonionic poly(ethylene oxide) surfactant (e.g., 5%) led to an improvement in macroporosity in abundance and in regularity with a slight enlargement in macropore sizes to 80-250 nm. Interestingly, a secondary, larger macropore system with parallel channels 500-1000 nm in size was generated when the synthesis was performed with moderately increasing the content of surfactant (10%), giving rise to an unprecedented trimodal meso-macro-macroporous structure. A uniform three-dimensional co-continuous macroporous structure with accessible wormhole-like mesoporous walls was synthesized by using the higher content of surfactants. This is a direct demonstration of tailoring the porous hierarchy of different lengths integrated in one solid body by fine-tuning the self-formation process and the participation of surfactant. The synthesized hierarchical titanium phosphates possess interesting optical and acidic properties, which should be significant for large application potential from catalysis and separation to electrochromic devices, fuel cells, and bioactive materials.

Synthesis and characterization of active ester-functionalized polypyrrole-silica nanoparticles: application to the covalent attachment of proteins.

Novel ester-functionalized polypyrrole-silica nanocomposite particles were prepared by oxidative copolymerization of pyrrole and N-succinimidyl ester pyrrole (50/50% initial concentrations), using FeCl3 in the presence of ultrafine silica nanoparticles (20 nm diameter). The N-succinimidyl ester pyrrole monomer was prepared in aqueous solution using 1-(2-carboxyethylpyrrole) and N-hydroxysuccinimide in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The resulting nanocomposites (N-succinimidyl ester polypyrrole-silica) are raspberry-shaped agglomerates of silica sol particles "glued" together by the insoluble poly(pyrrole-co-N-succinimidyl pyrrole). The N-succinimidyl ester polypyrrole-silica particles were characterized in terms of their size, density, copolymer content, and polydispersity. Scanning electron microscopy and disk centrifuge sedimentometry confirmed that the nanocomposite particles had narrow size distributions. X-ray photoelectron spectroscopy analysis indicated a silica-rich surface and a high surface concentration of N-succinimidyl ester groups. These nanoparticles exhibited good long-term dispersion stability. The chemical stability of the ester functions in aqueous media after several weeks of storage was monitored by FTIR spectroscopy. The functionalized nanocomposites were tested as bioadsorbents of human serum albumin (HSA). The very high amount of immobilized HSA determined by UV-visible spectroscopy is believed to be due to covalent binding. Incubation of the HSA-grafted nanocomposite with anti-HSA resulted in immediate flocculation, an indication that they are alternative candidates for visual diagnostic assays.