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Leukocyte Migration and Deformation in Collagen Gels and Microfabricated Constrictions
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Sáez PJ, Barbier L, Attia R, Thiam HR, Piel M, Vargas P.
Methods Mol Biol. - 1749 361-373 - doi: 10.1007/978-1-4939-7701-7_26. - 2018
In multicellular organisms, cell migration is a complex process. Examples of this are observed during cell motility in the interstitial space, full of extracellular matrix fibers, or when cells pass through endothelial layers to colonize or exit specific tissues. A common parameter for both situations is the fast adaptation of the cellular shape to their irregular landscape. In this chapter, we describe two methods to study cell migration in complex environments. The first one consists in a multichamber device for the visualization of cell haptotaxis toward the collagen-binding chemokine CCL21. This method is used to study cell migration as well as deformations during directed motility, as in the interstitial space. The second one consists in microfabricated channels connected to small constrictions. This procedure allows the study of cell deformations when single cells migrate through small holes and it is analogous to passage of cells through endothelial layers, resulting in a simplified system to study the mechanisms operating during transvasation. Both methods combined provide a powerful hub for the study of cell plasticity during migration in complex environments.
Leukocyte Migration and Deformation in Collagen Gels and Microfabricated Constrictions.
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Sáez PJ, Barbier L, Attia R, Thiam HR, Piel M, Vargas P
Methods Mol Biol. - 1749 361-373 - doi: 10.1007/978-1-4939-7701-7_26 - 2018
In multicellular organisms, cell migration is a complex process. Examples of this are observed during cell motility in the interstitial space, full of extracellular matrix fibers, or when cells pass through endothelial layers to colonize or exit specific tissues. A common parameter for both situations is the fast adaptation of the cellular shape to their irregular landscape. In this chapter, we describe two methods to study cell migration in complex environments. The first one consists in a multichamber device for the visualization of cell haptotaxis toward the collagen-binding chemokine CCL21. This method is used to study cell migration as well as deformations during directed motility, as in the interstitial space. The second one consists in microfabricated channels connected to small constrictions. This procedure allows the study of cell deformations when single cells migrate through small holes and it is analogous to passage of cells through endothelial layers, resulting in a simplified system to study the mechanisms operating during transvasation. Both methods combined provide a powerful hub for the study of cell plasticity during migration in complex environments.
Mixed Copolymer Adlayers Allowing Reversible Thermal Control of Single Cell Aspect Ratio.
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Dalier F1, Dubacheva GV1, Coniel M1, Zanchi D1,2, Galtayries A, Piel M, Marie E1, Tribet C1.
ACS Appl Mater Interfaces - 10(3) 2253-2258 - doi: 10.1021/acsami.7b18513. - 2018
Dynamic guidance of living cells is achieved by fine-tuning and spatiotemporal modulation on artificial polymer layers enabling reversible peptide display. Adjustment of surface composition and interactions is obtained by coadsorption of mixed poly(lysine) derivatives, grafted with either repellent PEG, RGD adhesion peptides, or T-responsive poly(N-isopropylacrylamide) strands. Deposition of mixed adlayers provides a straightforward mean to optimize complex substrates, which is here implemented to achieve (1) thermal control of ligand accessibility and (2) adjustment of relative adhesiveness between adjacent micropatterns, while preserving cell attachment during thermal cycles. The reversible polarization of HeLa cells along orthogonal stripes mimics guidance along natural matrices.
Diversification of human plasmacytoid predendritic cells in response to a single stimulus
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Alculumbre SG, Saint-André V, Di Domizio J, Vargas P, Sirven P, Bost P, Maurin M, Maiuri P, Wery M, Roman MS, Savey L, Touzot M, Terrier B, Saadoun D, Conrad C, Gilliet M, Morillon A, Soumelis V
Nat Immunol. - 19(1) 63-75 - doi: 10.1038/s41590-017-0012-z. - 2018
Innate immune cells adjust to microbial and inflammatory stimuli through a process termed environmental plasticity, which links a given individual stimulus to a unique activated state. Here, we report that activation of human plasmacytoid predendritic cells (pDCs) with a single microbial or cytokine stimulus triggers cell diversification into three stable subpopulations (P1-P3). P1-pDCs (PD-L1+CD80-) displayed a plasmacytoid morphology and specialization for type I interferon production. P3-pDCs (PD-L1-CD80+) adopted a dendritic morphology and adaptive immune functions. P2-pDCs (PD-L1+CD80+) displayed both innate and adaptive functions. Each subpopulation expressed a specific coding- and long-noncoding-RNA signature and was stable after secondary stimulation. P1-pDCs were detected in samples from patients with lupus or psoriasis. pDC diversification was independent of cell divisions or preexisting heterogeneity within steady-state pDCs but was controlled by a TNF autocrine and/or paracrine communication loop. Our findings reveal a novel mechanism for diversity and division of labor in innate immune cells.
A conductive hydrogel based on alginate and carbon nanotubes for probing microbial electroactivity
Laboratoire Colloïdes et Matériaux Divisés - Leopold Mottet, Domitille Le Cornec, a Jean-Marc Noe, Frederic Kanoufi, Brigitte Delord, Philippe Poulin, Jerome Bibettea and Nicolas Bremond
Soft Matter - 14 1434 - DOI: 10.1039/c7sm01929g - 2018
Some bacteria can act as catalysts to oxidize (or reduce) organic or inorganic matter with the potential
of generating electrical current. Despite their high value for sustainable energy, organic compound
production and bioremediation, a tool to probe the natural biodiversity and to select most efficient
microbes is still lacking. Compartmentalized cell culture is an ideal strategy for achieving such a goal but
the appropriate compartment allowing cell growth and electron exchange must be tailored. Here, we
develop a conductive composite hydrogel made of a double network of alginate and carbon nanotubes.
Homogeneous mixing of carbon nanotubes within the polyelectrolyte is obtained by a surfactant
assisted dispersion followed by a desorption step for triggering electrical conductivity. Dripping the
mixture in a gelling bath through simple extrusion or a double one allows the formation of either plain
hydrogel beads or liquid core hydrogel capsules. The process is shown to be compatible with the
bacterial culture (Geobacter sulfurreducens). Bacteria can indeed colonize the outer wall of plain beads
or the inner wall of the conductive capsules’ shell that function as an anode from which electrons
produced by the cells are collected.
Osmotic pressure in polyelectrolyte solutions: cell-model and bulk simulations
Laboratoire Colloïdes et Matériaux Divisés - Magnus Ullner, Khawla Qamhieh and Bernard Cabane
Soft Matter - 14 5832-5846 - https://doi.org/10.1039/C8SM00654G - 2018
The osmotic pressure of polyelectrolyte solutions as a function of concentration has been calculated by Monte Carlo simulations of a spherical cell model and by molecular dynamics simulations with periodic boundary conditions. The results for the coarse-grained polyelectrolyte model are in good agreement with experimental results for sodium polyacrylate and the cell model is validated by the bulk simulations. The cell model offers an alternative perspective on osmotic pressure and also forms a direct link to even simpler models in the form of the Poisson–Boltzmann approximation applied to cylindrical and spherical geometries. As a result, the non-monotonic behaviour of the osmotic coefficient seen in simulated salt-free solutions is shown not to rely on a transition between a dilute and semi-dilute regime, as is often suggested when the polyion is modelled as a linear flexible chain. The non-monotonic behaviour is better described as the combination of a finite-size effect and a double-layer effect. Parameters that represent the linear nature of the polyion, including an alternative to monomer concentration, make it possible to display a generalised behaviour of equivalent chains, at least at low concentrations. At high concentrations, local interactions become significant and the exact details of the model become important. The effects of added salt are also discussed and one conclusion is that the empirical additivity rule, treating the contributions from the polyelectrolyte and any salt separately, is a reasonable approximation, which justifies the study of salt-free solutions.
A conductive hydrogel based on alginate and carbon nanotubes for probing microbial electroactivity
Laboratoire Colloïdes et Matériaux Divisés - Leopold Mottet, Domitille Le Cornec, Jean-Marc Noel, Frederic Kanoufi, Brigitte Delord, Philippe Poulin, Jerome Bibette, and Nicolas Bremond
Soft Matter - 14 1434 - DOI: 10.1039/c7sm01929g - 2018
Some bacteria can act as catalysts to oxidize (or reduce) organic or inorganic matter with the potential of generating electrical current. Despite their high value for sustainable energy, organic compound production and bioremediation, a tool to probe the natural biodiversity and to select most efficient microbes is still lacking. Compartmentalized cell culture is an ideal strategy for achieving such a goal but the appropriate compartment allowing cell growth and electron exchange must be tailored. Here, we develop a conductive composite hydrogel made of a double network of alginate and carbon nanotubes. Homogeneous mixing of carbon nanotubes within the polyelectrolyte is obtained by a surfactant assisted dispersion followed by a desorption step for triggering electrical conductivity. Dripping the mixture in a gelling bath through simple extrusion or a double one allows the formation of either plain hydrogel beads or liquid core hydrogel capsules. The process is shown to be compatible with the bacterial culture (Geobacter sulfurreducens). Bacteria can indeed colonize the outer wall of plain beads or the inner wall of the conductive capsules' shell that function as an anode from which electrons produced by the cells are collected.

Micropipette-powered droplet based microfluidics
Laboratoire Colloïdes et Matériaux Divisés - Krzysztof Langer, Nicolas Bremonda, Laurent Boitard, Jean Baudry, and Jérôme Bibette
Biomicrofluidics - 12 044106 - https://doi.org/10.1063/1.5037795 - 2018
Droplet-based microfluidics, using water-in-oil emulsion droplets as micro-reactors, is becoming a widespread method for performing assays and especially in the cell biology field. Making a simple and highly portable system for creating emulsion droplets would help to continue the popularization of such a technique. Also, the ability to emulsify all the samples would strengthen this compartimenlization technique to handle samples with limited volume. Here, we propose a strategy of droplet formation that combines a classical flow-focusing microfluidic chip, which could be commercially available, with a standard laboratory adjustable micropipette. The micropipette is used as a negative pressure generator for controlling liquid flows. In that way, emulsification does neither require any electrical power supply nor a cumbersome device and functions with small liquid volumes. Droplet formation can be easily and safely performed in places with limited space, opening a wide range of applications especially in biological laboratory environments with higher level of safety regulations, i.e., BSL-3/4. Fortunately, the present methodology that involves small fluid volumes, and thus possible time dependent flow conditions, allows to minimize dead volume while keeping drops' size homogeneous. A physical characterization of droplet production and a model that describes the emulsion features, in terms of drop size and size distribution, are proposed for rationalizing the performances of the micropipette-powered emulsification process.
Adaptive response of yeast cells to triggered toxicity of phosphoribulokinase.
Laboratoire Colloïdes et Matériaux Divisés - Rouzeau C Dagkesamanskaya A Langer K Bibette J Baudry J Pompon D Anton-Leberre V
Res Microbiol - 169(6) 335-342 - DOI : 10.1016/j.resmic.2018.06.002 - 2018
Adjustment of plasmid copy number resulting from the balance between positive and negative impacts of borne synthetic genes, plays a critical role in the global efficiency of multistep metabolic engineering. Differential expression of co-expressed engineered genes is frequently observed depending on growth phases, metabolic status and triggered adjustments of plasmid copy numbers, constituting a dynamic process contributing to minimize global engineering burden. A yeast model involving plasmid based expression of phosphoribulokinase (PRKp), a key enzyme for the reconstruction of synthetic Calvin cycle, was designed to gain further insights into such a mechanism. A conditional PRK expression cassette was cloned either onto a low (ARS-CEN based) or a high (2-micron origin based) copy number plasmid using complementation of a trp1 genomic mutation as constant positive selection. Evolution of plasmid copy numbers, PRKp expressions, and cell growth rates were dynamically monitored following gene de-repression through external doxycycline concentration shifts. In the absence of RubisCO encoding gene permitting metabolic recycling, PRKp expression that led to depletion of ribulose phosphate, a critical metabolite for aromatic amino-acids biosynthesis, and accumulation of the dead-end diphosphate product contribute to toxicity. Triggered copy number adjustment was found to be a dynamic process depending both on plasmid types and levels of PRK induction. With the ARS-CEN plasmid, cell growth was abruptly affected only when level PRKp expression exceeded a threshold value. In contrast, a proportional relationship was observed with the 2-micron plasmid consistent with large copy number adjustments. Micro-compartment partitioning of bulk cultures by embedding individual cells into inverse culture medium/oil droplets, revealed the presence of slow and fast growing subpopulations that differ in relative proportions for low and high copy number plasmids.
Adaptive response of yeast cells to triggered toxicity of phosphoribulokinase.
Laboratoire Colloïdes et Matériaux Divisés - Rouzeau C Dagkesamanskaya A Langer K Bibette J Baudry J Pompon D Anton-Leberre V
Res Microbiol - 169(6) 335-342 - DOI : 10.1016/j.resmic.2018.06.002 - 2018
Adjustment of plasmid copy number resulting from the balance between positive and negative impacts of borne synthetic genes, plays a critical role in the global efficiency of multistep metabolic engineering. Differential expression of co-expressed engineered genes is frequently observed depending on growth phases, metabolic status and triggered adjustments of plasmid copy numbers, constituting a dynamic process contributing to minimize global engineering burden. A yeast model involving plasmid based expression of phosphoribulokinase (PRKp), a key enzyme for the reconstruction of synthetic Calvin cycle, was designed to gain further insights into such a mechanism. A conditional PRK expression cassette was cloned either onto a low (ARS-CEN based) or a high (2-micron origin based) copy number plasmid using complementation of a trp1 genomic mutation as constant positive selection. Evolution of plasmid copy numbers, PRKp expressions, and cell growth rates were dynamically monitored following gene de-repression through external doxycycline concentration shifts. In the absence of RubisCO encoding gene permitting metabolic recycling, PRKp expression that led to depletion of ribulose phosphate, a critical metabolite for aromatic amino-acids biosynthesis, and accumulation of the dead-end diphosphate product contribute to toxicity. Triggered copy number adjustment was found to be a dynamic process depending both on plasmid types and levels of PRK induction. With the ARS-CEN plasmid, cell growth was abruptly affected only when level PRKp expression exceeded a threshold value. In contrast, a proportional relationship was observed with the 2-micron plasmid consistent with large copy number adjustments. Micro-compartment partitioning of bulk cultures by embedding individual cells into inverse culture medium/oil droplets, revealed the presence of slow and fast growing subpopulations that differ in relative proportions for low and high copy number plasmids.
Use of photoswitchable fluorescent proteins for droplet-based microfluidic screening
Laboratoire Colloïdes et Matériaux Divisés - Adilya Dagkesamanskaya 1, Krzysztof Langer , Alexandra S Tauzin , Catherine Rouzeau , Delphine Lestrade , Gabrielle Potocki-Veronese , Laurent Boitard , Jérôme Bibette , Jean Baudry , Denis Pompon , Véronique Anton-Leberre
J Microbiol Methods. - 147 59-65 - DOI: 10.1016/j.mimet.2018.03.001 - 2018
Application of droplet-based microfluidics for the screening of microbial libraries is one of the important ongoing developments in functional genomics/metagenomics. In this article, we propose a new method that can be employed for high-throughput profiling of cell growth. It consists of light-driven labelling droplets that contain growing cells directly in a microfluidics observation chamber, followed by recovery of the labelled cells. This method is based on intracellular expression of green-to-red switchable fluorescent proteins. The proof of concept is established here for two commonly used biological models, E. coli and S. cerevisiae. Growth of cells in droplets was monitored under a microscope and, depending on the targeted phenotype, the fluorescence of selected droplets was switched from a "green" to a "red" state. Red fluorescent cells from labelled droplets were then successfully detected, sorted with the Fluorescence Activated Cell Sorting machine and recovered. Finally, the application of this method for different kind of screenings, in particular of metagenomic libraries, is discussed and this idea is validated by the analysis of a model mini-library.
Microfluidic magnetic fluidized bed for DNA analysis in continuous flow mode
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Iván Hernández-Neuta, IagoPereiro, Annika Ahlforda, DavideFerraro, Qiongdi Zhang, Jean-LouisViovy, Stéphanie Descroix, Mats Nilssona
ELSEVIER - 102 531-539 - https://doi.org/10.1016/j.bios.2017.11.064 - 2018
Magnetic solid phase substrates for biomolecule manipulation have become a valuable tool for simplification and automation of molecular biology protocols. However, the handling of magnetic particles inside microfluidic chips for miniaturized assays is often challenging due to inefficient mixing, aggregation, and the advanced instrumentation required for effective actuation. Here, we describe the use of a microfluidic magnetic fluidized bed approach that enables dynamic, highly efficient and simplified magnetic bead actuation for DNA analysis in a continuous flow platform with minimal technical requirements. We evaluate the performance of this approach by testing the efficiency of individual steps of a DNA assay based on padlock probes and rolling circle amplification. This assay comprises common nucleic acid analysis principles, such as hybridization, ligation, amplification and restriction digestion. We obtained efficiencies of up to 90% for these reactions with high throughput processing up to 120 μL of DNA dilution at flow rates ranging from 1 to 5 μL/min without compromising performance. The fluidized bed was 20–50% more efficient than a commercially available solution for microfluidic manipulation of magnetic beads. Moreover, to demonstrate the potential of this approach for integration into micro-total analysis systems, we optimized the production of a low-cost polymer based microarray and tested its analytical performance for integrated single-molecule digital read-out. Finally, we provide the proof-of-concept for a single-chamber microfluidic chip that combines the fluidized bed with the polymer microarray for a highly simplified and integrated magnetic bead-based DNA analyzer, with potential applications in diagnostics.
Dissecting Effects of Anti-cancer Drugs and Cancer-Associated Fibroblasts by On-Chip Reconstitution of Immunocompetent Tumor Microenvironments
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Marie Nguyen 1, Adele De Ninno, Arianna Mencattini, Fanny Mermet-Meillon, Giulia Fornabaio, Sophia S Evans, Mélissande Cossutta, Yasmine Khira, Weijing Han, Philémon Sirven, Floriane Pelon, Davide Di Giuseppe, Francesca Romana Berta
Cell Reports - 25(13) 3884-3893 - DOI: 10.1016/j.celrep.2018.12.015 - 2018
major challenge in cancer research is the complexity of the tumor microenvironment, which includes the host immunological setting. Inspired by the emerging technology of organ-on-chip, we achieved 3D co-cultures in microfluidic devices (integrating four cell populations: cancer, immune, endothelial, and fibroblasts) to reconstitute ex vivo a human tumor ecosystem (HER2+ breast cancer). We visualized and quantified the complex dynamics of this tumor-on-chip, in the absence or in the presence of the drug trastuzumab (Herceptin), a targeted antibody therapy directed against the HER2 receptor. We uncovered the capacity of the drug trastuzumab to specifically promote long cancer-immune interactions (>50 min), recapitulating an anti-tumoral ADCC (antibody-dependent cell-mediated cytotoxicity) immune response. Cancer-associated fibroblasts (CAFs) antagonized the effects of trastuzumab. These observations constitute a proof of concept that tumors-on-chip are powerful platforms to study ex vivo immunocompetent tumor microenvironments, to characterize ecosystem-level drug responses, and to dissect the roles of stromal components.
Microfluidic valve with zero dead volume and negligible back-flow for droplets handling
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - D.Ferraro, M.Serra, I. Ferrante, L.Viovy, S.Descroix
ELSEVIER - 25(13) 3884-3893 - https://doi.org/10.1016/j.snb.2017.12.002 - 2018
Droplet microfluidics allows the implementation of several biochemical and bioanalytical protocols in microfluidic devices. However, this integration typically requires the use of microfluidic valves, which allow the control of different operations on the same channels network. Unfortunately, the valves commercially available are almost exclusively suitable for-single phase flow, and only one valve system compatible with multi-phase systems has been presented so far in the literature. We present here a new pinch-like valve that withstands high pressures (tested up to 4 bar), allows droplet manipulation and combines the advantages of conventional pinch valves regarding the absence of dead volume, while making negligible the backflow generated (less than 2 nL). The conception and design of the presented valve, based on numerical simulations, is performed by an optimized microfabrication strategy, combining PDMS soft-lithography and 3D printing technologies. The valves had been tested regarding their mechanical stability and proving their capability of allowing droplets passing through them, without any perturbation. Finally, the very small back-flow volume generated during the actuation, has been exploited for various applications in droplets microfluidics, like droplet handling, droplet-on-demand generation and sorting.
Single-step immunoassays and microfluidic droplet operation: Towards a versatile approach for detection of amyloid-β peptide-based biomarkers of Alzheimer’s disease
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Thanh DucMai, Davide Ferraro, Nacéra Abouda, Renaud Renault, Marco Serra, Nguyet ThuyTran, Jean-LouisViovy, Claire Smadja, Stéphanie Descroix, MyriamTaverna
ELSEVIER - 255 2126-2135 - https://doi.org/10.1016/j.snb.2017.09.003 - 2018
In this study, single-step magnetic-beads based immunoassays was developed and for the first time adapted to microfluidic droplet operation for sequential determination of well-established amyloid-β peptide biomarkers (i.e. monomeric Aβ 1–42 and Aβ 1–40) for molecular diagnosis of Alzheimer’s disease (AD). With the developed sandwich assay protocol, the capture antibodies (i.e. monoclonal anti-Aβ antibodies that are specific for N-terminus of Aβ 1–42 and Aβ 1–40) grafted onto magnetic beads and the detection antibodies (i.e. beta amyloid 1–16 monoclonal antibody labeled with horseradish peroxidase) can simultaneously bind to monomeric β peptides in a single step. Aβ 1–42 and Aβ 1–40 in cerebrospinal fluid (CSF) samples were successfully detected using the developed batchwise immunoassay approach. With the aim at expanding the spectrum of traced Aβ peptides beyond Aβ 1–42 and Aβ 1–40 for more precise diagnostics of AD, the developed sandwich assay was for the first time coupled as a downstream module to peptide fractionation and collection using capillary isoelectric focusing. Demonstration of this methodological combination was carried out with Aβ 1–40, Aβ 1–40 and Aβ 5–40. The immunoassay in batchwise format was downscaled into a purpose-made microfluidic droplet platform allowing significant sample volume reduction and higher throughput. Using a series of 4 programmable magnetic tweezers to manipulate a train of nano-scale confined droplets containing magnetic beads, sample, washing and detection solutions, a sequence of 8 analyses could be realized within 45 min. This droplet based immunoassay was realized in a dedicated platform integrating an in-house-made light emitting diode (LED)-based fluorescent detector, replacing conventional microscopic setup so as to significantly reduce the construction cost and simplify the detection protocol. Using this microfluidic configuration coupled with LED-based detection, information on both Aβ 1–42 and Aβ 1–40 concentrations can be collected in a single sequence with less than 1 μL of sample.
Resolution improvement of 3D stereo-lithography through the direct laser trajectory programming: Application to microfluidic deterministic lateral displacement device
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Petra Juskova, Alexis Ollitrault, Marco Serra, Jean-Louis Viovy, Laurent Malaquin
Anal Chim Acta - 1000 239-247 - doi: 10.1016/j.aca.2017.11.062 - 2018
The vast majority of current microfluidic devices are produced using soft lithography, a technique with strong limitations regarding the fabrication of three-dimensional architectures. Additive manufacturing holds great promises to overcome these limitations, but conventional machines still lack the resolution required by most microfluidic applications. 3D printing machines based on two-photon lasers, in contrast, have the needed resolution but are too limited in speed and size of the global device. Here we demonstrate how the resolution of conventional stereolithographic machines can be improved by a direct programming of the laser path and can contribute to bridge the gap between the two above technologies, allowing the direct printing of features between 10 and 100 μm, corresponding to a large fraction of microfluidic applications. This strategy allows to achieve resolutions limited only by the physical size of the laser beam, decreasing by a factor at least 2× the size of the smallest features printable, and increasing their reproducibility by a factor 5. The approach was applied to produce an open microfluidic device with the reversible seal, integrating periodical patterns using the simple motifs, and validated by the fabrication of a deterministic lateral displacement particles sorting device. The sorting of polystyrene beads (diameter: 20 μm and 45 μm) was achieved with a specificity >95%, comparable with that achieved with arrays prepared by microlithography.
Resolution improvement of 3D stereo-lithography through the direct laser trajectory programming: Application to microfluidic deterministic lateral displacement device
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Petra Juskova, Alexis Ollitrault, Marco Serra, Jean-Louis Viovy, Laurent Malaquin
Anal Chim Acta - 1000 239-247 - doi: 10.1016/j.aca.2017.11.062 - 2018
The vast majority of current microfluidic devices are produced using soft lithography, a technique with strong limitations regarding the fabrication of three-dimensional architectures. Additive manufacturing holds great promises to overcome these limitations, but conventional machines still lack the resolution required by most microfluidic applications. 3D printing machines based on two-photon lasers, in contrast, have the needed resolution but are too limited in speed and size of the global device. Here we demonstrate how the resolution of conventional stereolithographic machines can be improved by a direct programming of the laser path and can contribute to bridge the gap between the two above technologies, allowing the direct printing of features between 10 and 100 μm, corresponding to a large fraction of microfluidic applications. This strategy allows to achieve resolutions limited only by the physical size of the laser beam, decreasing by a factor at least 2× the size of the smallest features printable, and increasing their reproducibility by a factor 5. The approach was applied to produce an open microfluidic device with the reversible seal, integrating periodical patterns using the simple motifs, and validated by the fabrication of a deterministic lateral displacement particles sorting device. The sorting of polystyrene beads (diameter: 20 μm and 45 μm) was achieved with a specificity >95%, comparable with that achieved with arrays prepared by microlithography.
On-a-chip tryptic digestion of transthyretin: a step toward an integrated microfluidic system for the follow-up of familial transthyretin amyloidosis
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Jeanne Bataille, Arthur Viodé, Iago Pereiro, Josiane P Lafleur, Fanny Varenne, Stéphanie Descroix, François Becher, Jörg P Kutter, Camille Roesch, Christian Poüs, Myriam Taverna, Antoine Pallandre, Claire Smadja, Isabelle Le Potier
Analyst - 143(5) 1077-1086 - DOI: 10.1039/c7an01737e - 2018
A microfluidic microreactor for trypsin mediated transthyretin (TTR) digestion has been developed as a step towards the elaboration of a fully integrated microdevice for the detection of a rare and disabling disease, the familial transthyretin amyloidosis (ATTR) which is related to specific TTR mutations. Therefore, an enzymatic microreactor coupled to an analytical step able to monitor the mutation of TTR on specific peptide fragments would allow an accurate monitoring of the treatment efficiency of ATTR. In this study, two types of immobilized trypsin microreactors have been investigated: a new miniaturized, microfluidic fluidized bed packed with trypsin functionalized magnetic particles (MPs), and a thiol-ene (TE) monolith-based chip. Their performances were first demonstrated with N-benzoyl-dl-arginine-4-nitroanilide hydrochloride BApNA, a low molecular weight substrate. High reaction yields (75.2%) have been reached within 0.6 min for the TE-based trypsin microreactor, while a lower yield (12.4%) was obtained for the micro-fluidized bed within a similar residence time. Transposition of the optimized conditions, developed with BApNA, to TTR digestion in the TE-based trypsin microreactor was successfully performed. We demonstrated that the TE-chip can achieve an efficient and reproducible digestion of TTR. This has been assessed by MS detection. In addition, TTR hydrolysis led to the production of a fragment of interest allowing the therapeutic follow-up of more than twenty possible ATTR mutations. High sequence coverage (90%), similar to those obtained with free trypsin, was achieved in a short time (2.4 min). Repeated experiments showed good reproducibility (RSD = 6.8%). These promising results open up the route for an innovative treatment follow-up dedicated to ATTR.
On-chip conductometric detection of short DNA sequences via electro-hydrodynamic aggregation
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - B. Venzac, M. L. Diakité, D. Herthnek, I. Cissé, U. Bockelmann, S. Descroix, L. Malaquinabcf and J.-L. Viovy
Analyst - 143 190-199 - https://doi.org/10.1039/C7AN00798A - 2018
Fluorescence measurement is the main technology for post-amplification DNA detection in automated systems. Direct electrical reading of DNA concentration in solution could be an interesting alternative to go toward more miniaturized or less expensive devices, in particular in the pathogen detection field. Here we present the detection of short bacterial biomarkers with a direct impedancemetric measurement, within solutions of amplified and elongated DNA sequences in a microchannel. This technology relies on the electrohydrodynamic instability occurring in solutions of long charged macromolecules in a strong electric field. This instability specifically induces the aggregation of long DNAs and triggers conductivity variations that can be monitored by on-contact conductometry. An innovative isothermal amplification and elongation strategy was developed, combining SDA and HRCA reactions, in order to yield long DNAs suitable to be detected by the above principle, from a dilute initial DNA target. In contrast with previous label-free detection methods, this new strategy is very robust to matrix effects, thanks to the unique molecular weight dependence of the instability, coupled with this specific DNA amplification strategy. We demonstrate the detection of a 1 pM gene sequence specific to Staphylococcus aureus, in a portable system.

A new biomimetic assay reveals the temporal role of matrix stiffening in cancer cell invasion
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Ralitza Staneva, Federica Burla, Gijsje H. Koenderink, Stéphanie Descroix, Danijela Matic Vignjevic, Youmna Attieh, and Marine Verhulse
Mol Biol Cell - 29(25) 2979–2988 - doi: 10.1091/mbc.E18-01-0068 - 2018
Tumor initiation and growth is associated with significant changes in the surrounding tissue. During carcinoma progression, a global stiffening of the extracellular matrix is observed and is interpreted as a signature of aggressive invasive tumors. However, it is still unknown whether this increase in matrix rigidity promotes invasion and whether this effect is constant along the course of invasion. Here we have developed a biomimetic in vitro assay that enabled us to address the question of the importance of tissue rigidity in the chronology of tumor invasion. Using low concentrations of the sugar threose, we can effectively stiffen reconstituted collagen I matrices and control the stiffening in time with no direct effect on residing cells. Our findings demonstrate that, depending on the timing of its stiffening, the extracellular matrix could either inhibit or promote cancer cell invasion and subsequent metastasis: while matrix stiffening after the onset of invasion promotes cancer cell migration and tumor spreading, stiff matrices encapsulate the tumor at an early stage and prevent cancer cell invasion. Our study suggests that adding a temporal dimension in in vitro models to analyze biological processes in four dimensions is necessary to fully capture their complexity.

584 publications.