Kevin Montagne

Programming an in vitro DNA oscillator using a molecular networking strategy

Living organisms perform and control complex behaviours by using webs of chemical reactions organized in precise networks. This powerful system concept, which is at the very core of biology, has recently become a new foundation for bioengineering. Remarkably, however, it is still extremely difficult to rationally create such network architectures in artificial, non‐living and well‐controlled settings. We introduce here a method for such a purpose, on the basis of standard DNA biochemistry. This approach is demonstrated by assembling de novo an efficient chemical oscillator: we encode the wiring of the corresponding network in the sequence of small DNA templates and obtain the predicted dynamics. Our results show that the rational cascading of standard elements opens the possibility to implement complex behaviours in vitro. Because of the simple and well‐controlled environment, the corresponding chemical network is easily amenable to quantitative mathematical analysis. These synthetic systems may thus accelerate our understanding of the underlying principles of biological dynamic modules.

The effect of substrate microtopography on focal adhesion maturation and actin organization via the RhoA/ROCK pathway

Recently, a growing number of reports have reported that micro- or nanoscale topography enhances cellular functions such as cell adhesion and stem cell differentiation, but the mechanisms responsible for this topography-mediated cell behavior are not fully understood. In this study, we examine the underlying processes and mechanisms behind specific topography-mediated cellular functions. Formation of focal adhesions (FA) was studied by culturing cells on different kinds of topographies, including a flat surface and surfaces with a micropatterned topography (2 μm lattice pattern with 3 μm intervals). We found that the formation and maturation of focal adhesions were highly dependent on the topography of the substrate although the shape, morphology and spreading of cells on the different substrates were not significantly affected. Focal adhesion maturation and actin polymerization were also promoted in cells cultured on the micropatterned substrate. These differences in cell adhesion led us to focus on the Rho GTPases, RhoA and downstream pathways since a number of reports have demonstrated that RhoA-activated cells have highly enhanced focal adhesions and actin activation such as polymerization. By inhibiting the Rho-associated kinase (ROCK) and downstream myosin II, we found that the FA formation, actin organization, and FAK phosphorylation were dramatically decreased. The topographical dependency of FA formation was also highly decreased. These results show that the FA formation and actin cytoskeleton organization of cells on the microtopography is regulated by the RhoA/ROCK pathway.

Micropit surfaces designed for accelerating osteogenic differentiation of murine mesenchymal stem cells via enhancing focal adhesion and actin polymerization

Recent reports demonstrate that enhanced focal adhesion (FA) between cells and the extracellular matrix (ECM) and intracellular actin polymerization (AP) upregulates cellular functions such as proliferation, stem-cell fate and differentiation. Purposed to accelerate osteogenic differentiation, enhancement of FAs and AP of cells was induced by adding a tailor-made micropit (tMP, 3 × 3 μm(2)) with different heights (2 or 4 μm). The tMP surface was examined for its differentiation efficiency using mouse mesenchymal stem cells, C3H10T1/2. Though the cell spreading area was not affected by the surface topography, cells on the tMP substrates had enhanced FAs which were significantly confined inside the micropits, increased actin polymerization and traction forces, and osteogenic differentiation. Further experiments with Y-27632 and Blebbistatin, which specifically regulate FA or AP functions, demonstrated that the tMP-induced acceleration of osteogenic differentiation was caused by the rho-associated, coiled-coil containing protein kinase (ROCK) and nonmuscle myosin II (NM II), which are key molecules of the RhoA/ROCK signaling pathway. The tMP is applicable as an osteo-active substrate for the instructive bone cell differentiation and population.

Towards single cell heat shock response by accurate control on thermal confinement with an on-chip microwire electrode

Metal electrodes with micron scale width enable the heating of less than a dozen cells in a confluent layer at predictable temperatures up to 85 °C with an accuracy of ±2 °C. Those performances were obtained by a preliminary robust temperature calibration based on biotin-rhodamine fluorescence and by controlling the temperature map on the substrate through thermal modeling. The temperature accuracy was proved by inducing the expression of heat shock proteins (HSP) in a few NIH-3T3 cells through a confined and precise temperature rise. Our device is therefore effective to locally induce a heat shock response with almost single-cell resolution. Furthermore, we show that cells heated at a higher temperature than the one of heat shock remain alive without producing HSP. Electrode deposition being one of the most common engineering processes, the fabrication of electrode arrays with a simple control circuit is clearly within reach for parallel testing. This should enable the study of several key mechanisms such as cell heat shock, death or signaling. In nanomedicine, controlled drug release by external stimuli such as for example temperature has attracted much attention. Our device could allow fast and efficient testing of thermoactivable drug delivery systems.

Laser sintering fabrication of three-dimensional tissue engineering scaffolds with a flow channel network

The fabrication of tissue engineering scaffolds for the reconstruction of highly oxygen-dependent inner organs is discussed. An additive manufacturing technology known as selective laser sintering was employed to fabricate a highly porous scaffold with an embedded flow channel network. A porogen leaching system was used to obtain high porosity. A prototype was developed using the biodegradable plastic polycaprolactone and sodium chloride as the porogen. A high porosity of 90% was successfully obtained. Micro x-ray CT observation was carried out to confirm that channels with a diameter of approximately 1 mm were generated without clogging. The amount of residual salt was 930 µg while the overall volume of the scaffold was 13 cm(3), and it was confirmed that the toxicity of the salt was negligible. The hydrophilization of the scaffold to improve cell adhesion on the scaffold is also discussed. Oxygen plasma ashing and hydrolysis with sodium hydroxide, typically employed to improve the hydrophilicity of plastic surfaces, were tested. The improvement of hydrophilicity was confirmed by an increase in water retention by the porous scaffold from 180% to 500%.

Combination of microwell structures and direct oxygenation enables efficient and size-regulated aggregate formation of an insulin-secreting pancreatic β-cell line.

Spherical three‐dimensional (3D) cellular aggregates are valuable for various applications such as regenerative medicine or cell‐based assays due to their stable and high functionality. However, previous methods to form aggregates have shown drawbacks, being labor‐intensive, showing low productivity per unit area or volume and difficulty to form homogeneous aggregates. We proposed a novel strategy based on oxygen‐permeable polydimethylsiloxane (PDMS) honeycomb microwell sheets, which can theoretically supply about 80 times as much oxygen as conventional polystyrene culture dishes, to produce recoverable aggregates in controllable sizes using mouse insulinoma cells (MIN6‐m9). In 48 hours of culture, the PDMS sheets produced aggregates whose diameters were strictly controlled (⋍32, 60, 90, 150 and 280 mm) even at an inoculum density eight times higher (8.0×105 cells/cm2) than that of normal confluent monolayers (1.0×105 cells/cm2). Measurement of the oxygen tension near the cell layer and glucose/lactate analysis clearly showed that cells exhibit aerobic respiration on the PDMS‐based culture system. Glucose‐responsive insulin secretion of the recovered aggregates showed that the aggregates around 90 mm in diameter secreted the largest amounts of insulin. This confirmed the advantages of 3D cellular organization and the existence of a suitable aggregate size, above which excess organization leads to a decreased metabolic response. These results demonstrated that this microwell‐based PDMS culture system provides a promising method to form size‐regulated and better functioning 3D cellular aggregates of various kinds of cells with a high yield per surface area.

Liver tissue engineering based on aggregate assembly: efficient formation of endothelialized rat hepatocyte aggregates and their immobilization with biodegradable fibres*

To realize long-term in vitro culture of hepatocytes at a high density while maintaining a high hepatic function for aggregate-based liver tissue engineering, we report here a novel culture method whereby endothelialized rat hepatocyte aggregates were formed using a PDMS microwell device and cultured in a perfusion bioreactor by introducing spacers between aggregates to improve oxygen and nutrient supply. Primary rat hepatocyte aggregates around 100 µm in diameter coated with human umbilical vein endothelial cells were spontaneously and quickly formed after 12 h of incubation, thanks to the continuous supply of oxygen by diffusion through the PDMS honeycomb microwell device. Then, the recovered endothelialized rat hepatocyte aggregates were mixed with biodegradable poly-l-lactic acid fibres in suspension and packed into a PDMS-based bioreactor. Perfusion culture of 7 days was successfully achieved with more than 73.8% cells retained in the bioreactor. As expected, the fibres acted as spacers between aggregates, which was evidenced from the enhanced albumin production and more spherical morphology compared with fibre-free packing. In summary, this study shows the advantages of using PDMS-based microwells to form heterotypic aggregates and also demonstrates the feasibility of spacing tissue elements for improving oxygen and nutrient supply to tissue engineering based on modular assembly.

Hydrostatic pressure decreases membrane fluidity and lipid desaturase expression in chondrocyte progenitor cells

Membrane biomechanical properties are critical in modulating nutrient and metabolite exchange as well as signal transduction. Biological membranes are predominantly composed of lipids, cholesterol and proteins, and their fluidity is tightly regulated by cholesterol and lipid desaturases. To determine whether such membrane fluidity regulation occurred in mammalian cells under pressure, we investigated the effects of pressure on membrane lipid order of mouse chondrogenic ATDC5 cells and desaturase gene expression. Hydrostatic pressure linearly increased membrane lipid packing and simultaneously repressed lipid desaturase gene expression. We also showed that cholesterol mimicked and cholesterol depletion reversed those effects, suggesting that desaturase gene expression was controlled by the membrane physical state itself. This study demonstrates a new effect of hydrostatic pressure on mammalian cells and may help to identify the molecular mechanisms involved in hydrostatic pressure sensing in chondrocytes.

Use of liposome encapsulated hemoglobin as an oxygen carrier for fetal and adult rat liver cell culture

Engineering liver tissue constructs with sufficient cell mass for transplantation implies culturing large numbers of hepatocytes in a reduced volume; however, providing sufficient oxygen to dense cell cultures is still not feasible using only conventional culture medium. Liposome-encapsulated hemoglobin (LEH), an oxygen-carrying blood substitute originally designed for short-term perfusion, may be a good candidate as an oxygen carrier to cultured liver cells. In this study, we investigated the feasibility of maintaining long term hepatocyte cultures using LEH. Primary fetal and adult rat liver cells were directly exposed to LEH for 6 to 14 days in static culture or in a perfused flat plate bioreactor. The functions and viability of adult rat hepatocytes exposed to LEH were not adversely affected in static monolayer culture and were even improved in the bioreactor. However, some cytotoxicity of LEH was observed with fetal rat liver cells after 4 days of culture. LEH, though a suitable oxygen carrier for long-term culture of mature hepatocytes, is not suitable in its present form for perfusing fetal hepatocyte cultures in direct contact with the liposomes; either the LEH will have to be made less toxic or a more sophisticated bioreactor that prevents the direct contact between hepatocytes and perfusates will have to be designed if fetal cells are to be used for liver tissue engineering.

Boosting functionality of synthetic DNA circuits with tailored deactivation

Molecular programming takes advantage of synthetic nucleic acid biochemistry to assemble networks of reactions, in vitro, with the double goal of better understanding cellular regulation and providing information-processing capabilities to man-made chemical systems. The function of molecular circuits is deeply related to their topological structure, but dynamical features (rate laws) also play a critical role. Here we introduce a mechanism to tune the nonlinearities associated with individual nodes of a synthetic network. This mechanism is based on programming deactivation laws using dedicated saturable pathways. We demonstrate this approach through the conversion of a single-node homoeostatic network into a bistable and reversible switch. Furthermore, we prove its generality by adding new functions to the library of reported man-made molecular devices: a system with three addressable bits of memory, and the first DNA-encoded excitable circuit. Specific saturable deactivation pathways thus greatly enrich the functional capability of a given circuit topology.