Cell traction forces in 3-D microenvironments

Las células son capaces de sentir y responder activamente frente a los estímulos mecánicos de su entorno. Los estímulos mecánicos que provienen de la matriz extracelular, tales como la rigidez, la topología de la superficie o la deformación, son traducidos en señales bioquímicas a través de las interacciones entre la célula y la matriz. Para poder sobrevivir y crecer las células necesitan adherirse y propagarse sobre el sustrato que las rodea. Una vez adheridas, las células generan fuerzas contráctiles a través de la interacción actina-miosina, ejerciendo de este modo tracción sobre el sustrato subyacente. Es por ello, que las fuerzas de tracción ejercidas por las células son reguladores críticos de la adhesión, la señalización y la función celular, y por tanto son muy importantes en numerosos procesos biológicos tales como la inflamación, la cicatrización de heridas, la angiogénesis e incluso la metástasis. Pese a su importancia, la medición de las fuerzas celulares en un contexto fisiológico así como entender su contribución en los procesos biológicos sigue siendo todavía un reto. Además, debido a que las interacciones célula-matriz varían considerablemente entre ambientes bidimensionales y tridimensionales, entender su influencia sobre las respuestas celulares normales y patológicas en sistemas tridimensionales es esencial para poder traducir de manera eficiente dichos conocimientos en terapias médicas. El principal objetivo de esta Tesis es, por tanto, el desarrollo de modelos computacionales enfocados al estudio de diferentes aspectos de las interacciones célula-matriz, que permitan entender mejor los fenómenos específicos y que sirvan como referencia para el desarrollo de nuevos experimentos y de técnicas de modelado in vitro. Además, todos los modelos y experimentos contenidos en esta tesis se centran en el estudio de células individuales. En primer lugar, debido a la complejidad y a las grandes diferencias que presentan con respecto a la migración celular colectiva, y en segundo lugar debido a la importancia que supone el estudio de la migración celular individual en procesos tan importantes como es la invasión de células tumorales. Además, debido a la relevancia que suponen fisiológicamente los entornos tridimensionales, en la mayoría de los modelos in silico desarrollados en esta Tesis, se han considerado aproximaciones tridimensionales para poder así imitar mejor las condiciones in vivo de células y tejidos.En primer lugar, se ha investigado la dinámica de unión de los sitios de adhesión célula-matriz, más en particular cómo las células transmiten las fuerzas a través de estas uniones a la matriz extracelular. Para ello, se ha desarrollado un modelo numérico mediante el uso del método de los elementos finitos [1]. En segundo lugar, se ha desarrollado un modelo in vitro para el estudio de las interacciones célula-matriz tanto a nivel celular como a nivel de tejido. En particular, se presentan diferentes dispositivos de microfluídica, los cuales están siendo utilizados en la actualidad para el estudio de diferentes procesos biológicos. Estos han sido utilizados para estudiar los procesos de formación de gradientes químicos a través de una matriz tridimensional [2]. Investigaciones recientes han indicado que las fuerzas de tracción celular son reguladores críticos de la invasión de las células tumorales, las cuales dependen en gran medida de las propiedades mecánicas tanto de las células como de la matriz que las rodea. Debido a que surge la necesidad de tener un conocimiento mucho más profundo sobre este mecanismo, la segunda parte de esta Tesis se ha centrado en el desarrollo de diferentes experimentos para cuantificar las fuerzas celulares, así como en el desarrollo de un modelo in silico basado en elementos finitos para reconstruir las fuerzas ejercidas por las células durante su migración, permitiendo de este modo estudiar la dependencia de las propiedades mecánicas de las células sobre la solución de fuerzas obtenida [3]. En resumen, una mejor comprensión de los mecanismos subyacentes a las interacciones célula-matriz, aportados en parte por la aparición de nuevas tecnologías para estudiar la mecánica celular a alta resolución espacial y temporal, no sólo resulta en una mejor comprensión del comportamiento de células normales, sino que también conduce al desarrollo de terapias novedosas para tratar enfermedades relacionadas con los defectos en las interacciones mecánicas celulares.<br /

Engineered environments for biomedical applications: anisotropic nanotopographies and microfluidic devices

During the last two decades micro- and nano-fabrication techniques originally developed for electronic engineering have directed their attention towards life sciences. The increase of analytical power of diagnostic devices and the creation of more biomimetic scaffolds have been strongly desired by these fields, in order to have a better insight into the complexity of physiological systems, while improving the ability to model them in vitro. Technological innovations worked to fill such a gap, but the integration of these fields of science is not progressing fast enough to satisfy the expectations. In this thesis I present novel devices which exploit the unique features of the micro- and nanoscale and, at the same time, match the requirements for successful application in biomedical research. Such biochips were used for optical detection of water-dispersed nanoparticles in microchannels, for highly controlled cell-patterning in closed microreactors, and for topography-mediated regulation of cell morphology and migration. Moreover, pilot experiments on the pre-clinical translation of micropatterned scaffolds in a rat model of peripheral nerve transaction were initiated and are ongoing. Given these results, the devices presented here have the potential to achieve clinical translation in a short/medium time, contributing to the improvement of biomedical technologies

Microfluidics Expanding the Frontiers of Microbial Ecology

Microfluidics has significantly contributed to the expansion of the frontiers of microbial ecology over the past decade by allowing researchers to observe the behaviors of microbes in highly controlled microenvironments, across scales from a single cell to mixed communities. Spatially and temporally varying distributions of organisms and chemical cues that mimic natural microbial habitats can now be established by exploiting physics at the micrometer scale and by incorporating structures with specific geometries and materials. In this article, we review applications of microfluidics that have resulted in insightful discoveries on fundamental aspects of microbial life, ranging from growth and sensing to cell-cell interactions and population dynamics. We anticipate that this flexible multidisciplinary technology will continue to facilitate discoveries regarding the ecology of microorganisms and help uncover strategies to control microbial processes such as biofilm formation and antibiotic resistance.National Science Foundation (U.S.) (Grant OCE-0744641-CAREER)National Science Foundation (U.S.) (Grant IOS-1120200)National Science Foundation (U.S.) (Grant CBET-1066566)National Science Foundation (U.S.) (Grant CBET-0966000)National Institutes of Health (U.S.) (NIH grant 1R01GM100473-0)Human Frontier Science Program (Strasbourg, France)Human Frontier Science Program (Strasbourg, France) (award RGY0089)Gordon and Betty Moore Foundation (Microbial Initiative Investigator Award

Keeping track of worm trackers

C. elegans is used extensively as a model system in the neurosciences due to its well defined nervous system. However, the seeming simplicity of this nervous system in anatomical structure and neuronal connectivity, at least compared to higher animals, underlies a rich diversity of behaviors. The usefulness of the worm in genome-wide mutagenesis or RNAi screens, where thousands of strains are assessed for phenotype, emphasizes the need for computational methods for automated parameterization of generated behaviors. In addition, behaviors can be modulated upon external cues like temperature, O2 and CO2 concentrations, mechanosensory and chemosensory inputs. Different machine vision tools have been developed to aid researchers in their efforts to inventory and characterize defined behavioral “outputs”. Here we aim at providing an overview of different worm-tracking packages or video analysis tools designed to quantify different aspects of locomotion such as the occurrence of directional changes (turns, omega bends), curvature of the sinusoidal shape (amplitude, body bend angles) and velocity (speed, backward or forward movement)

Lab-on-a-Chip Fabrication and Application

The necessity of on-site, fast, sensitive, and cheap complex laboratory analysis, associated with the advances in the microfabrication technologies and the microfluidics, made it possible for the creation of the innovative device lab-on-a-chip (LOC), by which we would be able to scale a single or multiple laboratory processes down to a chip format. The present book is dedicated to the LOC devices from two points of view: LOC fabrication and LOC application

Small

Screening functional phenotypes in small animals is important for genetics and drug discovery. Multiphase microfluidics has great potential for enhancing throughput but has been hampered by inefficient animal encapsulation and limited control over the animal's environment in droplets. Here, a highly efficient single-animal encapsulation unit, a liquid exchanger system for controlling the droplet chemical environment dynamically, and an automation scheme for the programming and robust execution of complex protocols are demonstrated. By careful use of interfacial forces, the liquid exchanger unit allows for adding and removing chemicals from a droplet and, therefore, generating chemical gradients inaccessible in previous multiphase systems. Using Caenorhabditis elegans as an example, it is demonstrated that these advances can serve to analyze dynamic phenotyping, such as behavior and neuronal activity, perform forward genetic screen, and are scalable to manipulate animals of different sizes. This platform paves the way for large-scale screens of complex dynamic phenotypes in small animals.P40 OD010440/CD/ODCDC CDC HHSUnited States/P40 OD010440/OD/NIH HHSUnited States/R01 AG056436/AG/NIA NIH HHSUnited States/R01NS096581/National Institute of Health/R01AG056436/National Institute of Health/ECCS-1542174/National Science Foundation/National Nanotechnology Coordinated Infrastructure/R01 NS096581/NS/NINDS NIH HHSUnited States/NIH R21NS117066/National Institute of Health/R21 NS117066/NS/NINDS NIH HHSUnited States

SURFACE ENABLED LAB-ON-A-CHIP (LOC) DEVICE FOR PROTEIN DETECTION AND SEPARATION

Sensitive and selective chemical/biological detection/analysis for proteins is essential for applications such as disease diagnosis, species phenotype identification, product quality control, and sample examination. Lab-on-a-chip (LOC) device provides advantages of fast analysis, reduced amount of sample requirements, and low cost, to magnificently facilitate protein detection research. Isoelectric focusing (IEF) is a strong and reliable electrophoretic technique capable of discerning proteins from complex mixtures based on the isoelectric point (pI) differences. It has experienced plenty of fruitful developments during previous decades which has given it the capability of performing with highly robust and reproducible analysis. This progress has made IEF devices an excellent tool for chemical/biological detection/analysis purposes. In recent years, the trends of simple instrument setting, rapid analysis, small sample requirement, and light labor intensity have inspired the LOC concept to be combined with IEF to evolve it into an “easily-handled chip with hours of analysis” from the earlier method of “working with big and heavy machines in a few days.” Although IEF is already a mature technique being applied, further LOC-IEF developments are still experiencing challenges related to its limitations such as miniaturizing the device scale without harming the resolving/discerning ability. With the facilitation of newly technologically advanced/improved fabrication tools, it is completely possible to address challenges and approach new limits of LOC-IEF. In this dissertation, a surface enabled printing technique, which can transfer liquid to a surface with prescribed patterns, was firstly introduced to IEF device fabrication. By employing surface enabled printing, a surface enabled IEF (sIEF) device running at a scale of 100 times smaller than those previously reported was designed and fabricated. Commercial carrier ampholytes (PharmalyteTM) with different pH range were engaged to generate a continuous pH gradient on sIEF device. Device design and optimized fabrication conditions were practically investigated; establishment of pH gradient was verified by fluorescent dyes; dependencies of electric field strength and carrier ampholytes concentration were systematically examined. To further optimize the sIEF system, dependencies of surface treatment and additive chemicals were explored. Fluorescent proteins and peptides were tested for the separation capability of sIEF. Finally, the well optimized sIEF system was used as a tool for real protein (hemoglobin variants and monoclonal antibody isoforms) separations. Hemoglobin variants test results revealed that sIEF is capable of separating amphoteric species with pI difference as small as 0.2. Monoclonal protein tests demonstrated the capability of sIEF to be a ready-to-use tool for protein structural change monitoring. In conclusion, this new sIEF approach has lower applied voltages, smaller sample requirements, a relatively quick fabrication process, and reusability, making it more attractive as a portable, user-friendly platform for qualitative protein detection and separation