Biological Cells Proliferation in Microwave Microsystems

International audienceThis paper presents the biological compatibility of a microwave analyzing microsystem of living cells through the indicator of cells proliferation. The cells under investigation correspond to adherent cells of Normal Rat Kidney (NRK). In a first time, both their adhesion and proliferation into the highfrequency-based micro-device have been successfully obtained. In a second step, microwave signals have been applied at different power levels. Experimental studies demonstrate that microwave power levels up to +8,6 dBm do not impact cells proliferation

Single step UV-photolithography fabrication of SU-8 honeycombs with microchannels for cells positioning on silicon oxide-based nanopillars

International audienceThis paper reports a novel technique to realize honeycomb arrangements of cell containers interconnected by microchannels using SU-8, a commonly used epoxy-based negative photoresist. These structures are fabricated using a single UV-photolithography exposure. With optimized process parameters, microchannels of various aspect ratios are produced. The cell containers and microchannels are used for the organization of axonal growth between neurons. Each container is centered on a nanopillar array promoting cell positioning. The proof of concept is given by the successful growth of interconnected PC12 cells. About 2-µm-high and 500-nm-wide silicon oxide-based nanopillars are produced by deep reactive ion etching using a photoresist mask. They are passivated by a 10-nm-thick silicon nitride layer. The resulting surface is biocompatible. The nanopillar arrays define areas for neurite and cell adhesion, while appropriately enhanced individual nanopillars can later be used for the recording of intracellular potentials. Next, the honeycomb structures are fabricated by a technique making use of a single photolithographic mask, a single SU-8 layer and standard equipment for stepper-based, optical projection lithography. Tuning the focus depth to negative values allows us to focus the reticle in the upper part of the SU-8 film, leading to an UV exposure imbalance between its upper and lower parts. As a consequence, pairs of neighboring SU-8 columns are connected in their upper parts by arches and separated in their lower parts by microchannels with aspect ratios depending on the column design parameters. Achieved microchannels widths range from 3 to 10 µm, while lengths extend from 5 to 30 µm for an SU-8 resist film thickness of 50 µm. After oxygen plasma treatment, PC12 cells in solution were cultured on the chip during one week. Cells settled on the whole chip and grew neurites through the SU-8 microchannels, thus setting up interconnections between all cell containers

Tuning the properties of silk fibroin biomaterial via chemical cross-linking

International audienceProtein-based biomaterials with innovative and controlled performance are being sought due to their unique characteristics for use in biomedical fields such as neural implants, drug delivery systems, cell-based therapies and soft tissue engineering. Here, we present a versatile approach for the synthesis of photo-crosslinkable fibroin silk biomaterial with highly tunable mechanical, chemical and biodegradation properties. Unlike the crystalline rich silk fibroin reported previously, the covalently cross-linked fibroin protein photoresist (FPP) via controlled light-induced radical grafting, allows generating a new amorphous biomaterial with tunable properties. It appears that the use of photo-reactive acrylate groups to cross-link FPP induces plasticity that can be tuned by changing the photoinitiator concentration of the film. Tensile strength measurements revealed that elasticity was higher for FPP UV-cross-linked materials with higher concentration of photoinitiator. FTIR and relative humidity measurements showed that hydrophilicity was higher for UV-cross-linked FPP. These materials display stiffness between 0.01–1.5 GPa and tensile strains up to 60%, covering a significant portion of the properties of native soft biomaterials. Besides, in vitro proteolytic degradation of the photocrosslinked FPP films demonstrate a tunable degradation rate over a period ranging from hours to weeks. Those biomaterials have been successfully micropatterned by photolithography techniques across several orders of magnitude (μm to cm) and a systematic study of direct patterning of the fibroin protein to form high fidelity and high-resolution structures has been reported. It was also shown that the fabricated protein features are well suited to cell adhesion. The development of protein-based material with controlled and tunable elasticity that can be easily photo-patterned into centimeter, micrometer and nanometer components will allow a wide range of applications in biomedical fields requesting a natural functional tissue

Multilevel (3D) microfluidic technology for an innovative magnetic cell separation and couting platform

International audienceCurrently, the technique for the quantitative detection of cells is flow cytometry. This technique has the advantage of being sensitive and reliable but is expensive, time consuming and not suited to both routine screening and point‐of‐care diagnostics. Miniaturized cell separation devices offer many advantages such as the use of small volumes, portability and low cost.We propose a new concept of device which, by combining 3D fluid engineering and localized magnetic actuation, enables the full integration of cell tagging, magnetic separation and cell counting in a single device. The labs on chip are manufactured by laminating commercially available photosensitive dry film that fits microfluidic requirements and gives the possibility to build easily 3D microfluidic systems.We show we can tag efficiently THP1 monocytes and subsequently sort them through magnetic trapping on integrated micro-coils. The separation efficiency is studied at different flow rates. Cell counting capacity, evaluated by using non‐ faradic impedance spectroscopy revealed that the cell trapping is selective, depending on the specific antibody grafted and quantitative with the range of detection being 1000 to 30000 infected cells. This range of detection is consistent with the targeted application

Deep plasma etching of Parylene C patterns for biomedical applications

We report on the plasma etching of thick Parylene C (~25 µm) in order to define flexible implantable probes for neural applications. Parylene C is a transparent polymer that presents with high biocompatibility, flexibility and chemical inertness, and has gained increased attention over the years in the biomedical field. In the manufacturing process, highly defined structuration steps of Parylene C are essential, but techniques based on laser, scalpel and wet etching have shown to be unsuitable for properly cut structures, i.e. with good dimensions control and without residues. Here, for the first time, negative resist (BPN) coating followed by RIE-ICP are used in order to pattern Parylene C-based structures, with a clean cut, vertical profile, fast etching rate (~0.8 µm/min) and conservation of device biocompatibility

Multilevel (3D) microfluidic technology for an innovative magnetic cell separation and couting platform

International audienceCurrently, the technique for the quantitative detection of cells is flow cytometry. This technique has the advantage of being sensitive and reliable but is expensive, time consuming and not suited to both routine screening and point‐of‐care diagnostics. Miniaturized cell separation devices offer many advantages such as the use of small volumes, portability and low cost.We propose a new concept of device which, by combining 3D fluid engineering and localized magnetic actuation, enables the full integration of cell tagging, magnetic separation and cell counting in a single device. The labs on chip are manufactured by laminating commercially available photosensitive dry film that fits microfluidic requirements and gives the possibility to build easily 3D microfluidic systems.We show we can tag efficiently THP1 monocytes and subsequently sort them through magnetic trapping on integrated micro-coils. The separation efficiency is studied at different flow rates. Cell counting capacity, evaluated by using non‐ faradic impedance spectroscopy revealed that the cell trapping is selective, depending on the specific antibody grafted and quantitative with the range of detection being 1000 to 30000 infected cells. This range of detection is consistent with the targeted application

Low-cost multilevel microchannel lab on chip: DF- 1000 series dry film photoresist as a promising enabler

International audienceWe demonstrate the use of a novel dry film photoresist DF-1000 series for the fabrication of multilevel microfluidic devices by combining a standard lithography technique and lamination technology. The optimization of the technological process enables achievement of high aspect ratio structures: 7 : 1 for free standing structures and 5 : 1 for channel structures. We proved that DF films feature a low autofluorescence level, similar to that of the SU-8 resist and compatible with most lab-on-a-chip applications. The chemical stability against aggressive solvents was also investigated. Last but not least, the non-cytotoxic effect according to ISO 10993-5 on the development of L-929 mouse fibroblast cells was established. Ultimately, we showed that this low-cost material combined with multilevel lamination and UV-lithography techniques allowed the fabrication of 3D microfluidic mixers and opened the way to perform microfluidics in three dimensions

Deep plasma etching of Parylene C patterns for biomedical applications

International audienceWe report on the plasma etching of thick (~23µm) Parylene C structures. Parylene C is a transparent polymer that benefits from high biocompatibility, flexibility and chemical inertness, and has gained increased attention over the years in the biomedical field. In the manufacturing process, highly defined structuration steps of Parylene C are essential, but techniques based on laser, scalpel and wet etching have shown to be unsuitable for properly cut structures. Plasma etching remains nowadays the most widespread option, though fast etching rate, lack of residues and high aspect ratios are still hard to achieve. To overcome these issues, the selection of both mask material and plasma conditions is crucial. Here, three masks-metal, positive and negative photoresists-are tested as stencils, and several plasma parameters are briefly studied in order to obtain the highest etching rate while maintaining good coverage. We showed that increasing the RF power up to a considerable 2800W while maintaining a moderate physical contribution (bias power, pressure, temperature), is optimal in the achievement of fast PaC etching without inducing thermal stress. Besides, the addition of a short fluorinated plasma in the midst of the process is shown to alleviate residues. For the first time, negative photoresist Intervia Bump Plating (BPN) coating followed by ICP 1-RIE 2 are used in order to pattern Parylene C-based structures, with a clean cut, vertical profile and fast etching rate (~0.87±0.06 µm/min) and a selectivity of 0.5. This solution was carried out to release unitary Parylene-based neural probes from a silicon wafer. Finally, cytotoxicity assays on these neural implants were performed to make sure that no trace of mask or stripper residues would jeopardize device biocompatibility

Adipose Stem Cells (ASCs) isolation by on-chip pre-treatment of biological samples

International audienceThis study presents the development of an original 2-steps Lab-on-chip that aims at isolating Adipose Stem Cells (ASCs), cells of considerable interest for regenerative medicine, from complex biological samples. Based on hydrodynamic filtration, the device pre-isolates ASCs by removing cells with a diameter below 10µm such as red blood cells or lymphocytes

MISFET-based biosensing interface for neurons guided growth and neuronal electrical activities recording

International audienceA hybrid circuit of a transistor-based chip was implemented and characterized for the neuronal electrical activity recording. The integration of microfluidic architectures was developed to control precisely neurites outgrowth and form topologically defined and stable neural networks. Individual neural cells from rat retinae and Lymnaea stagnalis snails were immobilized on gates regions of Metal Insulator Semiconductor Field Effect Transistors (MISFET). Neuronal orientation was achieved in both cases but neuronal action potentials were only recorded in the Lymnaea stagnalis case. They were successfully triggered and inhibited by implementing a picrotoxin-GABA – picrotoxin injection protocol, exhibiting a direct influence of picrotoxin on the "spike type" action potential waveform. The implementation of the whole process of neuronal culture and subsequent activity monitoring constitutes a proof-of-principle experiment for the development of neuroelectronic systems for signal processing studies adapted to low-density neuronal cultures