Christophe Provin

A method for the design of 3D scaffolds for high-density cell attachment and determination of optimum perfusion culture conditions.

The application of in vitro cultured cells in tissue engineering or drug screening, aimed at complex soft tissues such as liver, requires in vivo physiological function of the cultured cells. For this purpose, the scaffold in which cells are cultured should provide a microenvironment similar to an in vivo one with a three-dimensional extracellular matrix, a high supply capacity of O(2) and nutrients, and high cell density. In this paper, we propose a method to design (1) the geometry of the scaffold, with a surface/volume ratio optimized to allow high-density (5 x 10(7)cells/mL) cell culture and (2) culture conditions that will supply optimal quantities of oxygen and nutrients. CFD modeling of mass transport was used to determine the shear stress as well as O(2) and glucose metabolism in the scaffold (20 mm width-35 mm length) for various flow rates. Validation of the model was done through comparison with flow resistance and micro-PIV experiments. CFD analysis showed the maximum metabolic rate densities for this scaffold are 6.04 x 10(-3)mol/s/m(3) for O(2) at 0.71 mL/min and 1.91 x 10(-2)mol/s/m(3) for glucose at 0.35 mL/min.

Complex ceramic-polymer composite microparts made by microstereolithography

In this paper we deal with the use of microstereolithography (/spl mu/SL) to produce solid freeform objects from computer-assisted-design files. The /spl mu/SL process is derived from stereolithography, and it is based on the photopolymerization through a dynamic mask generator of successive layers of photocurable resin, which permits us to produce accurate micro-objects with high aspect ratio and curved surfaces (due to the layer-by-layer nature of the process). This technology is extended to the manufacture of ceramic-polymer composite parts. To achieve this, we add dispersed alumina powder (at a volumic percentage of 24%) and a visible photoinitiator to a low viscosity diacrylate resin. The objects we made present interesting properties for microrobotic or microfluidic applications.

Micellar extraction of europium (III) by a bolaform extractant and parent compounds derived from 5-pyrazolone

Abstract Micellar ultrafiltration is used to determine the extraction of europium (III) by a series of micelle-solubilized extractants derived from 5-pyrazolone. The extractants, solubilized in CTAB (cetyltrimethylammonium bromide) micelles, have either a single complexing site: 1-phenyl-3-methyl-4-benzoyl-5-pyrazolone (HPMBP), 1-phenyl-3-methyl-4-lauroyl-5-pyrazolone (HPMLP) or they are bis-ligands which can be called ‘bolaform’ extractants: 1,6-bis(1′-phenyl-3′-methyl-5′-pyrazolone-4′-)-1,6-hexanedione (HP-4-PH), 1,12-bis(1′-phenyl-3′-methyl-5′-pyrazolone-4′-)-1,12-dodecanedione (HP-10-PH). The yield of extraction is measured as a function of the pH and of the extractant/metal ratio. The efficiencies of the extractants are in the order HP-10-PH>HPMLP>HPMBP and the complex stabilities are larger than in standard solvent extraction, the former ligand giving a yield of europium extraction close to 100% at pH 2. A theoretical model is used to determine the stoichiometries of the extracted complexes, which are then compared with the situations encountered in standard solvent extraction. For the single site complexing agents the complexes formed involved three ligand molecules (L) for one metal. For the bolaform extractants the complex stoichiometries are found to be either EuL 2 H (case of HP-4-PH) or Eu 2 L 3 (case of HP-10-PH). The stoichiometries and stabilities of the complexes are discussed in relation with the confinement effect offered by the micellar particles and the effect of the local concentrations. The association of a CTA + surfactant molecule to the complex, which was suggested by previous works to ensure neutrality, appears here to be unlikely.

Low O2 metabolism of HepG2 cells cultured at high density in a 3D microstructured scaffold.

Among the features of in vivo liver cells that are rarely mimicked in vitro, especially in microchips, is the very high cell density. In this study, we have cultured HepG2 in a plate-type PDMS scaffold with a three-dimensional ordered microstructure optimally designed to allow cells to attach at a density of 108 cells/mL. After the first step of static open culture, the scaffold was sealed to simulate the in vivo oxygen supply, which is supplied only through the perfusion of medium. The oxygen consumption rate at various flow rates was measured. An average maximal cellular oxygen consumption rate of 3.4 × 10−17 mol/s/cell was found, which is much lower than previously reported values for hepatocytes. Nevertheless, the oxygen concentration in the bulk stream was not the limiting factor. It has been further confirmed by the reported numerical model that the mass transport resistance on the surface of a cell that limits the oxygen supply to the cell. These results further emphasize that access to a sufficient quantity of oxygen, especially through the diffusion-limited layer on the surface of a cell, is very important for the metabolism of hepatocytes at such a high density.

An Integrated Microfluidic System for Manganese Anomaly Detection Based on Chemiluminescence: Description and Practical Use to Discover Hydrothermal Plumes Near the Okinawa Trough

Underwater hydrothermal vents draw a lot of interest as they are the shelter for unique ecosystems, besides being a possible new opportunity for ore mining. Manganese (Mn) is found at a high concentration in hydrothermal vents, which make it possible to use this metal ion as a tracer to detect and evaluate new hydrothermal sources. Here we present a miniaturized and integrated microfluidic system for the detection of Mn in deep-sea environment, called the integrated in situ analyzer for Mn2+ (IISA-Mn). The detection system is based on the chemiluminescence reaction of Mn contained in the seawater sample with a luminol-based reagent, which offers a high sensitivity. This system is composed of a microdevice for mixing and reaction, a pumping unit, several valving units, and a photomultiplier (PMT) detector. The system is able to detect Mn concentration above 280 nM in seawater, and gives a quite linear response until 500 nM. It is also proven to be able to work continuously during the 8 h of an actual remotely operated vehicle (ROV) dive. This system has led to the discovery of a previously unknown hydrothermal site in the Okinawa Trough.

Micro-scale rapid prototyping by stereolithography

A new process of microstereolithography to manufacture freeform solid three-dimensional micro-components with outer dimensions in the millimetre size range is presented. It corresponds to a layered micromachining, specially suited to rapid prototyping at the micro scale. Different kinds of materials can be employed, like pure polymers or alumina-based composites. The micromachine is mainly composed of a liquid crystal display as a dynamic mask generator and of a broad-band visible light source. The manufacture of complex three dimensional microparts demonstrates the possibilities of the process specially when concerned with composite alumina-based microstructures. Its availability to realise pure sintered alumina microcomponents is also shown.

Reaction–diffusion phenomena in a PDMS matrix can modify its topography

Various reagents and solvents can be absorbed into polydimethylsiloxane (PDMS), which may be a concern for many applications. We hypothesize that these absorbed reagents can also react with each other within the elastomer matrix. Here we demonstrate this phenomenon and use it as a means to physically modify the surface topography of the PDMS by generating wrinkles or pores.

Integrated In Situ Analyzer for Manganese (IISA-Mn) for Deep Sea Environment

Many metal ions that can be found at trace or ultratrace level in ocean are coming from deep-sea hydrothermal vents. In particular, manganese (Mn2+) can be assimilated by phytoplankton and so can be found in all the food chain. It is then of interest to be able to detect manganese to determine its spatial distribution and/or to find new hydrothermal sources. Usually, determination of manganese concentration is done by sampling seawater in bottles, followed by analysis in lab onboard a ship or on the ground. Major drawbacks of that method are that only a few samples can be taken during each dive and that there is a risk of contamination of the sample. The use of microfabrication techniques to produce an in-situ microanalyzer that can be installed in an autonomous underwater vehicles (AUVs) or remotely operated vehicles (ROVs) can allow a higher sampling rate, with experiments over a longer time, a reduced risk of contamination. We propose here a microdevice designed for in situ detection of manganese ion in deep-sea environment. Detection is based on chemiluminescence of luminol triggered by the H2O2 decomposition catalyzed by manganese ion. The set-up is composed of pressure-proof micropumps for handling of the reagents, a 6-way valve for samples introduction, a PDMS (poly(dimethylsiloxane)) microchip made by soft lithography, and a photomultiplier for photon detection. The limit of detection of this system is about 10 nanomolar at the moment.

New process for manufacturing ceramic microfluidic devices for microreactor and bioanalytical applications

The advances of the past few years in microreactors have demonstrated that microchips have numerous significant advantages with respect to cost, safety, throughput, kinetics and scale-up [1-3]. The whole aspect of heat management, enabling mass and heat transfer to be extremely rapid, leads to a higher level of reaction control and reactant manipulation at any one point within the chip.

A Microfluidic Diffusion Cell for Fast and Easy Percutaneous Absorption Assays

ABSTRACTPurposePercutaneous absorption assays of molecules for pharmaceutical and cosmetology purposes are important to determine the bioavailability of new compounds, once topically applied. The current method of choice is to measure the rate of diffusion through excised human skin using a diffusion cell. This method however entails significant drawbacks such as scarce availability and poor reproducibility of the sample, low sampling rate, and tedious assay setup.MethodsThe objective of the present work is to propose an alternative method that overcomes these issues by integrating an experimental model of the skin (artificial stratum corneum) and online optical sensors into a microfluidic device.ResultsThe measurement of the diffusion profile followed by the calculation of the permeability coefficients and time lag were performed on seven different molecules and obtained data positively fit with those available from literature on human skin penetration. The coating of the lipid mixture to generate the artificial stratum corneum also proved robust and reproducible. The results show that the proposed device is able to give fast, real-time, accurate, and reproducible data in a user-friendly manner, and can be produced at a large scale.ConclusionThese assets should help both the cosmetics and pharmaceutics fields where the skin is the target or a pathway of a formulated compound, by allowing more candidate molecules or formulations to be assessed during the various stages of their development.