Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 40
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Biomed Opt Express ; 10(11): 5862-5876, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31799052

RESUMO

This work describes the implementation of a compact system allowing measurement of blood flow velocity using laser Doppler velocimetry in situ. The compact setup uses an optical fiber acting as an emitter and receptor of the signal. The signal is then recovered by a photodiode and processed using a spectrum analyzer. The prototype was successfully tested to measure microbead suspension and whole blood flow velocities in a fluidic chip. Fibers with hemispherical lenses with three different radius of curvature were investigated. This simple yet precise setup would enable the insertion of the fiber via a medical catheter to monitor blood flow velocity in non superficial vessels where previous reported techniques cannot be implemented.

2.
Brain Res Bull ; 152: 202-211, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31348979

RESUMO

The development of cellular microenvironments suitable for neural tissue engineering purposes involves a plethora of research fields ranging from cell biology to biochemistry, neurosciences, physics, nanotechnology, mechanobiology. In the last two decades, this multi-disciplinary activity has led to the emergence of numerous strategies to create architectures capable of reproducing the topological, biochemical and mechanical properties of the extracellular matrix present in the central (CNS) and peripheral nervous system (PNS). Some of these approaches have succeeded in inducing the functional recovery of damaged areas in the CNS and the PNS to address the current lack of effective medical treatments for this type of injury. In this review, we analyze recent developments in the realization of two-dimensional and three-dimensional neuronal scaffolds following either top-down or bottom-up approaches. After providing an overview of the different fabrication techniques employed for tailoring the biomaterials, we draw on specific examples to describe the major features of the developed approaches. We then conclude with prospective proof of concept studies on guiding scaffolds and regenerative models on macro-scale brain implants targeting neural regeneration.

3.
PLoS One ; 14(5): e0217227, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31120960

RESUMO

Growing multicellular spheroids recapitulate many features of expanding microtumours, and therefore they are an attractive system for biomechanical studies. Here, we report an original approach to measure and characterize the forces exerted by proliferating multicellular spheroids. As force sensors, we used high aspect ratio PDMS pillars arranged as a ring that supports a growing breast tumour cell spheroid. After optical imaging and determination of the force application zones, we combined 3D reconstruction of the shape of each deformed PDMS pillar with the finite element method to extract the forces responsible for the experimental observation. We found that the force exerted by growing spheroids ranges between 100nN and 300nN. Moreover, the exerted force was dependent on the pillar stiffness and increased over time with spheroid growth.


Assuntos
Neoplasias da Mama/patologia , Técnicas de Cultura de Células/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Esferoides Celulares/patologia , Feminino , Humanos , Estresse Mecânico , Análise Serial de Tecidos
4.
Nano Lett ; 18(10): 6326-6333, 2018 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-30232897

RESUMO

In vivo, immune cells migrate through a wide variety of tissues, including confined and constricting environments. Deciphering how cells apply forces when infiltrating narrow areas is a critical issue that requires innovative experimental procedures. To reveal the distribution and dynamics of the forces of cells migrating in confined environments, we designed a device combining microchannels of controlled dimensions with integrated deformable micropillars serving as sensors of nanoscale subcellular forces. First, a specific process composed of two steps of photolithography and dry etching was tuned to obtain micrometric pillars of controlled stiffness and dimensions inside microchannels. Second, an image-analysis workflow was developed to automatically evaluate the amplitude and direction of the forces applied on the micropillars by migrating cells. Using this workflow, we show that this microdevice is a sensor of forces with a limit of detection down to 64 pN. Third, by recording pillar movements during the migration of macrophages inside the confining microchannels, we reveal that macrophages bent the pillars with typical forces of 0.3 nN and applied higher forces at the cell edges than around their nuclei. When the degree of confinement was increased, we found that forces were redirected from inward to outward. By providing a microdevice that allows the analysis of force direction and force magnitude developed by confined cells, our work paves the way for investigating the mechanical behavior of cells migrating though 3D constricted environments.


Assuntos
Técnicas de Cultura de Células , Núcleo Celular/química , Dispositivos Lab-On-A-Chip , Macrófagos/química , Técnicas Biossensoriais/métodos , Adesão Celular/genética , Movimento Celular/genética , Núcleo Celular/genética , Microambiente Celular/genética , Voluntários Saudáveis , Humanos , Fenômenos Mecânicos , Monócitos/química
5.
PLoS One ; 13(8): e0202531, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30138342

RESUMO

Microcontact printing has become a versatile soft lithography technique used to produce molecular micro- and nano-patterns consisting of a large range of different biomolecules. Despite intensive research over the last decade and numerous applications in the fields of biosensors, microarrays and biomedical applications, the large-scale implementation of microcontact printing is still an issue. It is hindered by the stamp-inking step that is critical to ensure a reproducible and uniform transfer of inked molecules over large areas. This is particularly important when addressing application such as cell microarray manufacturing, which are currently used for a wide range of analytical and pharmaceutical applications. In this paper, we present a large-scale and multiplexed microcontact printing process of extracellular matrix proteins for the fabrication of cell microarrays. We have developed a microfluidic inking approach combined with a magnetic clamping technology that can be adapted to most standard substrates used in biology. We have demonstrated a significant improvement of homogeneity of printed protein patterns on surfaces larger than 1 cm2 through the control of both the flow rate and the wetting mechanism of the stamp surface during microfluidic inking. Thanks to the reproducibility and integration capabilities provided by microfluidics, we have achieved the printing of three different adhesion proteins in one-step transfer. Selective cell adhesion and cell shape adaptation on the produced patterns were observed, showing the suitability of this approach for producing on-demand large-scale cell microarrays.


Assuntos
Proteínas da Matriz Extracelular/isolamento & purificação , Técnicas Analíticas Microfluídicas/métodos , Impressão/instrumentação , Análise Serial de Tecidos/instrumentação , Técnicas Biossensoriais , Adesão Celular/genética , Forma Celular/genética , Proteínas da Matriz Extracelular/química
6.
J Vis Exp ; (136)2018 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-29985327

RESUMO

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction forces have been well-characterized, but there is a lack of techniques allowing the measurement of the protrusion forces exerted by cells orthogonally to their substrate. We designed an experimental setup to measure the protrusion forces exerted by adherent cells on their substrate. Cells plated on a compliant Formvar sheet deform this substrate and the resulting topography is mapped by atomic force microscopy (AFM) at the nanometer scale. Force values are then extracted from an analysis of the deformation profile based on the geometry of the protrusive cellular structures. Hence, the forces exerted by the individual protruding units of a living cell can be measured over time. This technique will enable the study of force generation and its regulation in the many cellular processes involving protrusion. Here, we describe its application to measure the protrusive forces generated by podosomes formed by human macrophages.


Assuntos
Fenômenos Fisiológicos Celulares/fisiologia , Macrófagos/fisiologia , Microscopia de Força Atômica/métodos , Podossomos/fisiologia , Animais , Humanos
7.
Methods Mol Biol ; 1771: 83-95, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29633206

RESUMO

Biomolecule microarrays are generally produced by conventional microarrayer, i.e., by contact or inkjet printing. Microcontact printing represents an alternative way of deposition of biomolecules on solid supports but even if various biomolecules have been successfully microcontact printed, the production of biomolecule microarrays in routine by microcontact printing remains a challenging task and needs an effective, fast, robust, and low-cost automation process. Here, we describe the production of biomolecule microarrays composed of extracellular matrix protein for the fabrication of cell microarrays by using an automated microcontact printing device. Large scale cell microarrays can be reproducibly obtained by this method.


Assuntos
Impressão Tridimensional , Análise Serial de Tecidos/métodos , Técnicas de Cultura de Células , Materiais Revestidos Biocompatíveis , Proteínas da Matriz Extracelular , Análise Serial de Tecidos/instrumentação
8.
Stem Cell Res Ther ; 8(1): 253, 2017 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-29116017

RESUMO

BACKGROUND: The adult brain is unable to regenerate itself sufficiently after large injuries. Therefore, hopes rely on therapies using neural stem cell or biomaterial transplantation to sustain brain reconstruction. The aim of the present study was to evaluate the improvement in sensorimotor recovery brought about by human primary adult neural stem cells (hNSCs) in combination with bio-implants. METHODS: hNSCs were pre-seeded on implants micropatterned for neurite guidance and inserted intracerebrally 2 weeks after a primary motor cortex lesion in rats. Long-term behaviour was significantly improved after hNSC implants versus cell engraftment in the grip strength test. MRI and immunohistological studies were conducted to elucidate the underlying mechanisms of neuro-implant integration. RESULTS: hNSC implants promoted tissue reconstruction and limited hemispheric atrophy and glial scar expansion. After 3 months, grafted hNSCs were detected on implants and expressed mature neuronal markers (NeuN, MAP2, SMI312). They also migrated over a short distance to the reconstructed tissues and to the peri-lesional tissues, where 26% integrated as mature neurons. Newly formed host neural progenitors (nestin, DCX) colonized the implants, notably in the presence of hNSCs, and participated in tissue reconstruction. The microstructured bio-implants sustained the guided maturation of both grafted hNSCs and endogenous progenitors. CONCLUSIONS: These immunohistological results are coherent with and could explain the late improvement observed in sensorimotor recovery. These findings provide novel insights into the regenerative potential of primary adult hNSCs combined with microstructured implants.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Células-Tronco Neurais/fisiologia , Células-Tronco Neurais/transplante , Regeneração/fisiologia , Diferenciação Celular/fisiologia , Humanos , Engenharia Tecidual
9.
Biomed Microdevices ; 19(3): 60, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28677098

RESUMO

We present a new strategy for fabricating a silicon nanopore device allowing straightforward fluidic integration and electrical as well as optical monitoring. The device presents nanopores of diameters 10 nm to 160 nm, and could therefore be used to obtain solvent-free free-standing lipid bilayers from small unilamellar vesicles (SUV) or large unilamellar vesicles (LUV). The silicon chip fabrication process only requires front side processing of a silicon-on-insulator (SOI) substrate. A polydimethylsiloxane (PDMS) microfluidic interface is assembled on the silicon chip for fluidic handling and electrical addressing. We detail the electrical specifications of our device and some perspectives showing that the use of an SOI substrate is a convenient way to reduce the electrical noise in a silicon nanopore device without the need of a photolitographic patterned passivation layer. We then demonstrate simultaneous electrical and optical monitoring by capturing negatively charged fluorescent nanoparticles. Finally, in the perspective of solvent-free free-standing lipid bilayers, we show that incubation of SUV results in a drastic increase of the device electrical resistance, which is likely due to the formation of a free-standing lipid bilayer sealing the nanopores. Graphical abstract ᅟ.


Assuntos
Corantes Fluorescentes/química , Dispositivos Lab-On-A-Chip , Bicamadas Lipídicas/química , Nanopartículas/química , Nanoporos , Imagem Óptica , Dimetilpolisiloxanos/química , Impedância Elétrica
10.
Small ; 13(27)2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28558136

RESUMO

The realization of 3D architectures for the study of cell growth, proliferation, and differentiation is a task of fundamental importance for both technological and biological communities involved in the development of biomimetic cell culture environments. Here we report the fabrication of 3D freestanding scaffolds, realized by multiphoton direct laser writing and seeded with neuroblastoma cells, and their multitechnique characterization using advanced 3D fluorescence imaging approaches. The high accuracy of the fabrication process (≈200 nm) allows a much finer control of the micro- and nanoscale features compared to other 3D printing technologies based on fused deposition modeling, inkjet printing, selective laser sintering, or polyjet technology. Scanning electron microscopy (SEM) provides detailed insights about the morphology of both cells and cellular interconnections around the 3D architecture. On the other hand, the nature of the seeding in the inner core of the 3D scaffold, inaccessible by conventional SEM imaging, is unveiled by light sheet fluorescence microscopy and multiphoton confocal imaging highlighting an optimal cell colonization both around and within the 3D scaffold as well as the formation of long neuritic extensions. The results open appealing scenarios for the use of the developed 3D fabrication/3D imaging protocols in several neuroscientific contexts.


Assuntos
Materiais Biocompatíveis/química , Imageamento Tridimensional/métodos , Polímeros/química , Engenharia Tecidual/métodos , Tecidos Suporte/química , Linhagem Celular Tumoral , Humanos , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência
11.
ACS Nano ; 11(4): 4028-4040, 2017 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-28355484

RESUMO

Determining how cells generate and transduce mechanical forces at the nanoscale is a major technical challenge for the understanding of numerous physiological and pathological processes. Podosomes are submicrometer cell structures with a columnar F-actin core surrounded by a ring of adhesion proteins, which possess the singular ability to protrude into and probe the extracellular matrix. Using protrusion force microscopy, we have previously shown that single podosomes produce local nanoscale protrusions on the extracellular environment. However, how cellular forces are distributed to allow this protruding mechanism is still unknown. To investigate the molecular machinery of protrusion force generation, we performed mechanical simulations and developed quantitative image analyses of nanoscale architectural and mechanical measurements. First, in silico modeling showed that the deformations of the substrate made by podosomes require protrusion forces to be balanced by local traction forces at the immediate core periphery where the adhesion ring is located. Second, we showed that three-ring proteins are required for actin polymerization and protrusion force generation. Third, using DONALD, a 3D nanoscopy technique that provides 20 nm isotropic localization precision, we related force generation to the molecular extension of talin within the podosome ring, which requires vinculin and paxillin, indicating that the ring sustains mechanical tension. Our work demonstrates that the ring is a site of tension, balancing protrusion at the core. This local coupling of opposing forces forms the basis of protrusion and reveals the podosome as a nanoscale autonomous force generator.


Assuntos
Podossomos/química , Actinas/química , Actinas/metabolismo , Fenômenos Biomecânicos , Adesão Celular , Células Cultivadas , Simulação por Computador , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Mecanotransdução Celular , Monócitos/citologia , Monócitos/metabolismo , Nanoestruturas/química , Tamanho da Partícula , Paxilina/química , Paxilina/metabolismo , Podossomos/ultraestrutura , Propriedades de Superfície , Talina/química , Talina/metabolismo , Vinculina/química , Vinculina/metabolismo
12.
Neural Plast ; 2017: 2545736, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29391951

RESUMO

Stroke represents the first cause of adult acquired disability. Spontaneous recovery, dependent on endogenous neurogenesis, allows for limited recovery in 50% of patients who remain functionally dependent despite physiotherapy. Here, we propose a review of novel drug therapies with strong potential in the clinic. We will also discuss new avenues of stem cell therapy in patients with a cerebral lesion. A promising future for the development of efficient drugs to enhance functional recovery after stroke seems evident. These drugs will have to prove their efficacy also in severely affected patients. The efficacy of stem cell engraftment has been demonstrated but will have to prove its potential in restoring tissue function for the massive brain lesions that are most debilitating. New answers may lay in biomaterials, a steadily growing field. Biomaterials should ideally resemble lesioned brain structures in architecture and must be proven to increase functional reconnections within host tissue before clinical testing.


Assuntos
Plasticidade Neuronal , Transplante de Células-Tronco , Reabilitação do Acidente Vascular Cerebral/métodos , Acidente Vascular Cerebral/terapia , Animais , Materiais Biocompatíveis , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Humanos , Nanotecnologia , Fármacos Neuroprotetores , Recuperação de Função Fisiológica , Acidente Vascular Cerebral/tratamento farmacológico
13.
Methods ; 94: 75-84, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26342257

RESUMO

Podosomes are submicron adhesive and mechanosensitive structures formed by macrophages, dendritic cells and osteoclasts that are capable of protruding into the extracellular environment. Built of an F-actin core surrounded by an adhesion ring, podosomes assemble in a network interconnected by acto-myosin cables. They have been shown to display spatiotemporal instability as well as protrusion force oscillations. To analyse the entire population of these unstable structures, we have designed an automated multi-particle tracking adapted to both topographical and fluorescence data. Here we describe in detail this approach and report the measurements of individual and collective characteristics of podosome ensembles, providing an integrated picture of their activity from the complementary angles of organisation, dynamics, mobility and mechanics. We believe that this will lead to a comprehensive view of podosome collective behaviour and deepen our knowledge about the significance of mechanosensing mediated by protrusive structures.


Assuntos
Macrófagos/fisiologia , Podossomos/fisiologia , Células Cultivadas , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Macrófagos/ultraestrutura , Microscopia de Força Atômica , Microscopia de Fluorescência , Podossomos/ultraestrutura
14.
PLoS One ; 10(9): e0138308, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26398500

RESUMO

Manganese-enhanced MRI (MEMRI) has been described as a powerful tool to depict the architecture of neuronal circuits. In this study we investigated the potential use of in vivo MRI detection of manganese for tracing neuronal projections from the primary motor cortex (M1) in healthy marmosets (Callithrix Jacchus). We determined the optimal dose of manganese chloride (MnCl2) among 800, 400, 40 and 8 nmol that led to manganese-induced hyperintensity furthest from the injection site, as specific to the corticospinal tract as possible, and that would not induce motor deficit. A commonly available 3T human clinical MRI scanner and human knee coil were used to follow hyperintensity in the corticospinal tract 24h after injection. A statistical parametric map of seven marmosets injected with the chosen dose, 8 nmol, showed the corticospinal tract and M1 connectivity with the basal ganglia, substantia nigra and thalamus. Safety was determined for the lowest dose that did not induce dexterity and grip strength deficit, and no behavioral effects could be seen in marmosets who received multiple injections of manganese one month apart. In conclusion, our study shows for the first time in marmosets, a reliable and reproducible way to perform longitudinal ME-MRI experiments to observe the integrity of the marmoset corticospinal tract on a clinical 3T MRI scanner.


Assuntos
Manganês/farmacologia , Técnicas de Rastreamento Neuroanatômico/métodos , Tratos Piramidais/fisiologia , Animais , Comportamento Animal , Encéfalo/efeitos dos fármacos , Encéfalo/fisiologia , Callithrix , Cloretos/administração & dosagem , Cloretos/farmacologia , Estudos de Viabilidade , Feminino , Processamento de Imagem Assistida por Computador , Imagem por Ressonância Magnética/instrumentação , Masculino , Compostos de Manganês/administração & dosagem , Compostos de Manganês/farmacologia , Tratos Piramidais/efeitos dos fármacos , Estatística como Assunto
15.
ACS Nano ; 9(4): 3800-13, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25791988

RESUMO

Podosomes are mechanosensitive adhesion cell structures that are capable of applying protrusive forces onto the extracellular environment. We have recently developed a method dedicated to the evaluation of the nanoscale forces that podosomes generate to protrude into the extracellular matrix. It consists in measuring by atomic force microscopy (AFM) the nanometer deformations produced by macrophages on a compliant Formvar membrane and has been called protrusion force microscopy (PFM). Here we perform time-lapse PFM experiments and investigate spatial correlations of force dynamics between podosome pairs. We use an automated procedure based on finite element simulations that extends the analysis of PFM experimental data to take into account podosome architecture and organization. We show that protrusion force varies in a synchronous manner for podosome first neighbors, a result that correlates with phase synchrony of core F-actin temporal oscillations. This dynamic spatial coordination between podosomes suggests a short-range interaction that regulates their mechanical activity.


Assuntos
Actinas/metabolismo , Fenômenos Mecânicos , Podossomos/metabolismo , Actinas/química , Fenômenos Biomecânicos , Análise de Elementos Finitos , Humanos , Macrófagos/citologia , Microscopia de Força Atômica , Modelos Moleculares , Monócitos/citologia , Conformação Proteica
16.
Adv Mater ; 27(7): 1268-73, 2015 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-25410279

RESUMO

An innovative technique is proposed for the precise and scalable placement of 1D nanostructures in an affordable manner. This approach combines the dielectrophoresis phenomenon and capillary assembly to successfully align thousands of single nanowires at specific locations at the wafer. The nanowires are selectively trapped by taking advantage of the material--specific frequence dependence.


Assuntos
Nanofios/química , Catálise , Eletrodos , Eletroforese , Ouro/química , Silício/química
17.
Nat Commun ; 5: 5343, 2014 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-25385672

RESUMO

Podosomes are adhesion structures formed in monocyte-derived cells. They are F-actin-rich columns perpendicular to the substrate surrounded by a ring of integrins. Here, to measure podosome protrusive forces, we designed an innovative experimental setup named protrusion force microscopy (PFM), which consists in measuring by atomic force microscopy the deformation induced by living cells onto a compliant Formvar sheet. By quantifying the heights of protrusions made by podosomes onto Formvar sheets, we estimate that a single podosome generates a protrusion force that increases with the stiffness of the substratum, which is a hallmark of mechanosensing activity. We show that the protrusive force generated at podosomes oscillates with a constant period and requires combined actomyosin contraction and actin polymerization. Finally, we elaborate a model to explain the mechanical and oscillatory activities of podosomes. Thus, PFM shows that podosomes are mechanosensing cell structures exerting a protrusive force.


Assuntos
Relógios Biológicos/fisiologia , Macrófagos/fisiologia , Mecanotransdução Celular/fisiologia , Microscopia de Força Atômica/métodos , Podossomos/fisiologia , Actinas/fisiologia , Actinas/ultraestrutura , Extensões da Superfície Celular/fisiologia , Extensões da Superfície Celular/ultraestrutura , Células Cultivadas , Humanos , Macrófagos/citologia , Macrófagos/ultraestrutura , Microscopia Eletrônica de Varredura , Modelos Biológicos , Simulação de Dinâmica Molecular , Podossomos/ultraestrutura
18.
Lab Chip ; 14(13): 2344-53, 2014 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-24836927

RESUMO

We report the design, fabrication and evaluation of an array of microdevices composed of high aspect ratio PDMS pillars, dedicated to the study of tumour spheroid mechanical properties. The principle of the microdevice is to confine a spheroid within a circle of micropillars acting as peripheral flexible force sensors. We present a technological process for fabricating high aspect ratio micropillars (300 µm high) with tunable feature dimensions (diameter and spacing) enabling production of flexible PDMS pillars with a height comparable to spheroid sizes. This represents an upscale of 10 along the vertical direction in comparison to more conventional PDMS pillar force sensors devoted to single cell studies, while maintaining their force sensitivity in the same order of magnitude. We present a method for keeping these very high aspect ratio PDMS pillars stable and straight in liquid solution. We demonstrate that microfabricated devices are biocompatible and adapted to long-term spheroid growth. Finally, we show that the spheroid interaction with the micropillars' surface is dependent on PDMS cellular adhesiveness. Time-lapse recordings of growth-induced micropillars' bending coupled with a software program to automatically detect and analyse micropillar displacements are presented. The use of these microdevices as force microsensors opens new prospects in the fields of tissue mechanics and pharmacological drug screening.


Assuntos
Técnicas de Cultura de Células , Neoplasias do Colo/patologia , Dimetilpolisiloxanos/química , Técnicas Analíticas Microfluídicas , Esferoides Celulares/patologia , Análise Serial de Tecidos , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Humanos , Teste de Materiais/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Esferoides Celulares/metabolismo , Análise Serial de Tecidos/instrumentação , Análise Serial de Tecidos/métodos
19.
Langmuir ; 30(11): 3132-41, 2014 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-24568716

RESUMO

There is an increasing interest to express and study membrane proteins in vitro. New techniques to produce and insert functional membrane proteins into planar lipid bilayers have to be developed. In this work, we produce a tethered lipid bilayer membrane (tBLM) to provide sufficient space for the incorporation of the integral membrane protein (IMP) Aquaporin Z (AqpZ) between the tBLM and the surface of the sensor. We use a gold (Au)-coated sensor surface compatible with mechanical sensing using a quartz crystal microbalance with dissipation monitoring (QCM-D) or optical sensing using the surface plasmon resonance (SPR) method. tBLM is produced by vesicle fusion onto a thin gold film, using phospholipid-polyethylene glycol (PEG) as a spacer. Lipid vesicles are composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethyleneglycol)-2000-N-[3-(2-pyridyldithio)propionate], so-called DSPE-PEG-PDP, at different molar ratios (respectively, 99.5/0.5, 97.5/2.5, and 95/5 mol %), and tBLM formation is characterized using QCM-D, SPR, and atomic force technology (AFM). We demonstrate that tBLM can be produced on the gold surface after rupture of the vesicles using an α helical (AH) peptide, derived from hepatitis C virus NS5A protein, to assist the fusion process. A cell-free expression system producing the E. coli integral membrane protein Aquaporin Z (AqpZ) is directly incubated onto the tBLMs for expression and insertion of the IMP at the upper side of tBLMs. The incorporation of AqpZ into bilayers is monitored by QCM-D and compared to a control experiment (without plasmid in the cell-free expression system). We demonstrate that an IMP such as AqpZ, produced by a cell-free expression system without any protein purification, can be incorporated into an engineered tBLM preassembled at the surface of a gold-coated sensor.


Assuntos
Aquaporinas/biossíntese , Aquaporinas/genética , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Ouro/química , Bicamadas Lipídicas/química , Aquaporinas/química , Técnicas Biossensoriais , Membrana Celular/química , Proteínas de Escherichia coli/química , Polietilenoglicóis/química , Propionatos/química , Piridinas/química , Propriedades de Superfície
20.
Macromol Biosci ; 13(11): 1546-55, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24039002

RESUMO

To investigate the respective roles of topography and cell/cell interactions in the development of a guided neuronal network on an engineered surface, micropatterned PDMS substrates were generated with different microgrooves geometry and investigated for the influence of cell density on neurite outgrowth and alignment. Through this systematic investigation, using a human neuronal stem cell line, the rules of neuronal network development and guiding could be learned. The results show that when cell density is increased the influence on neuritic outgrowth and alignment is very different for the various grooves geometries. The data emphasized the competition, in neurite development, between physical cues brought by surface topographical features and cell to cell communications. These results can be of particular interest for designing functional neuronal networks with a controlled architecture.


Assuntos
Rede Nervosa/anatomia & histologia , Células-Tronco Neurais/citologia , Neuritos/ultraestrutura , Engenharia Tecidual , Adesão Celular/efeitos dos fármacos , Comunicação Celular/efeitos dos fármacos , Contagem de Células , Diferenciação Celular , Linhagem Celular , Dimetilpolisiloxanos/química , Dimetilpolisiloxanos/farmacologia , Humanos , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/fisiologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/fisiologia , Neuritos/efeitos dos fármacos , Nylons/química , Nylons/farmacologia , Propriedades de Superfície , Tecidos Suporte
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA