26 research outputs found

    Techniques to stimulate and interrogate cell–cell adhesion mechanics

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    Cell–cell adhesions maintain the mechanical integrity of multicellular tissues and have recently been found to act as mechanotransducers, translating mechanical cues into biochemical signals. Mechanotransduction studies have primarily focused on focal adhesions, sites of cell-substrate attachment. These studies leverage technical advances in devices and systems interfacing with living cells through cell–extracellular matrix adhesions. As reports of aberrant signal transduction originating from mutations in cell–cell adhesion molecules are being increasingly associated with disease states, growing attention is being paid to this intercellular signaling hub. Along with this renewed focus, new requirements arise for the interrogation and stimulation of cell–cell adhesive junctions. This review covers established experimental techniques for stimulation and interrogation of cell–cell adhesion from cell pairs to monolayers

    Lab-On-Chip for Ex-Vivo Study of Bio-Mechanical-Chemical Behavior of Tip Growing Plant Cells

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    This thesis presents design, modeling, fabrication, and testing of different Lab-on-chip (LOC) devices to study static and dynamic behavior of pollen tubes in bio-mechanical-chemical environments. The main components of microfluidic platform include microfluidic network for manipulation, trapping and growing of a series of pollen tubes in a controlled environment, actuating channels in order to introduce chemicals and drugs toward the pollen tube, microstructural elements such as microgaps and microcantilevers to provide Ex-Vivo environment for characterizing static and dynamic responses of pollen tubes. A Lab-On-Chip (LOC), called, TipChip was developed as a flexible platform that can simplify sophisticated functions such as chemical reactions, drug development, by integrating them within a single micro-device. The configuration of the microfluidic network was developed in such a way that it allows observation under chemical or mechanical manipulation of multiple pollen tubes. The growth of pollen tubes under different flow rates and geometrical dimensions of microfluidic network has been studied and the challenges have been identified. The microfluidic platform design was enhanced to deal with the challenges by adapting the dimensions of the microfluidic network and the inlet flow. It provides identical growth environments for growing pollen tubes along each microchannel and improves the performance of microfluidic device, through varying the dimensions and geometries of the microfluidic network. The thesis identifies the static response of pollen tube to chemical stimulation which was used to determine the role of a few of the growth regulators such as sucrose and calcium ions as they regulate tube turgor pressure and cell wall mechanical properties of pollen tube. New experimental platforms were fabricated to treat locally the pollen tube at the tip in order to characterize its static response to local treatment in reorienting the growth direction. The device is also used to locally stimulate the cylindrical region of pollen tube. Using these LOC devices we attempted to answer some questions regarding the role of regulators in pollen tube growth. The thesis explores in detail the dynamic growth of pollen tube in normal condition and also under chemical stimulation. Waveform analysis is employed in order to extract primary and secondary oscillation frequencies of pollen tube as significant indicators of dynamic growth of pollen tube. The dynamic response of pollen tubes is implemented as a whole-plant cell sensor for toxicity detection in order to detect toxic materials in concentration-based manner. Aluminum ions were tested as the toxic substance. The degree of toxicity was defined by measuring the reduction in growth rate as well as peak oscillation frequencies in the case of static and dynamic response of pollen tube, respectively. The thesis addresses the quantification of mechanical properties of pollen tube cell wall using the Bending LOC (BLOC) platform. The flexural rigidity of the pollen tube and the Young’s modulus of the cell wall are estimated through finite element modeling of the observed fluid-structure interaction. The thesis also explores the feasibility of studying the pollen tube response to the mechanical stimulation. The microfluidic device also enables integrating mechanical force obstructing pollen tube growth in order to characterize the interaction of pollen tube and mechanical structures which are similar to the in-vivo interaction between a pollen tube and the growth matrix during the course of growth toward the ovule. The behavior of the pollen tube while passing through microgap was also explored in detail. The deflection of microgap under growth force and the changes in diameter of the pollen tube under reaction force from microgap were evaluated. This part explores the role of mechanical forces in bursting the pollen tube tip which could explain the contribution of mechanical signal in the bursting of tube near the vicinity of the ovule. In addition, the configuration of microgap enabled the estimation of the maximum invasive force exerted by pollen tube. Thus, the proposed microfluidic platform is highly suitable for cellular analysis, pollen tube biology and detection of toxicity

    Novel miniaturised and highly versatile biomechatronic platforms for the characterisation of melanoma cancer cells

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    There has been an increasing demand to acquire highly sensitive devices that are able to detect and characterize cancer at a single cell level. Despite the moderate progress in this field, the majority of approaches failed to reach cell characterization with optimal sensitivity and specificity. Accordingly, in this study highly sensitive, miniaturized-biomechatronic platforms have been modeled, designed, optimized, microfabricated, and characterized, which can be used to detect and differentiate various stages of melanoma cancer cells. The melanoma cell has been chosen as a legitimate cancer model, where electrophysiological and analytical expression of cell-membrane potential have been derived, and cellular contractile force has been obtained through a correlation with micromechanical deflections of a miniaturized cantilever beam. The main objectives of this study are in fourfold: (1) to quantify cell-membrane potential, (2) correlate cellular biophysics to respective contractile force of a cell in association with various stages of the melanoma disease, (3) examine the morphology of each stage of melanoma, and (4) arrive at a relation that would interrelate stage of the disease, cellular contractile force, and cellular electrophysiology based on conducted in vitro experimental findings. Various well-characterized melanoma cancer cell lines, with varying degrees of genetic complexities have been utilized. In this study, two-miniaturized-versatile-biomechatronic platforms have been developed to extract the electrophysiology of cells, and cellular mechanics (mechanobiology). The former platform consists of a microfluidic module, and stimulating and recording array of electrodes patterned on a glass substrate, forming multi-electrode arrays (MEAs), whereas the latter system consists of a microcantilever-based biosensor with an embedded Wheatstone bridge, and a microfluidic module. Furthermore, in support of this work main objectives, dedicated microelectronics together with customized software have been attained to functionalize, and empower the two-biomechatronic platforms. The bio-mechatronic system performance has been tested throughout a sufficient number of in vitro experiments.Open Acces

    Cell traction forces in 3-D microenvironments

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    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 /

    AN INTEGRATED MICROSYSTEM FOR BACTERIAL BIOFILM DETECTION AND TREATMENT

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    Bacterial biofilms cause severe infections in clinical fields and contamination problems in environmental facilities. Due to the unique complex structure of biofilms that comprise diverse polysaccharides and bacteria, traditional antibiotic therapies require a thousand times higher concentration compared to non-biofilm associated infections. The early detection of biofilms, before their structures are fully established in a given host/environment, is critical in order to eradicate them effectively. Also, the development of a new innovative biofilm treatment method that can be utilized with a low dose of antibiotic would be extremely important to the medical community. In this dissertation, a biofilm sensor and a new biofilm treatment method were independently developed to detect and treat biofilm communities, respectively. Furthermore, an integrated microsystem was demonstrated as a single platform of the sensor with the treatment method. The sensor was based on the surface acoustic wave (SAW) detection mechanism, which isn extremely sensitive for biofilm monitoring (hundreds of bacterial population detection limit) and consumes very low power (~100 µW). A piezoelectric ZnO layer fabricated by a pulsed laser deposition process was a key material to induce homogeneous acoustic waves. Reliable operation of the sensor was achieved using an Al2O3 film as a passivation layer over the sensor to protect ZnO degradation from the growth media. The sensor successfully demonstrated real-time monitoring of biofilm growth. The new biofilm treatment was developed based on the principles of the bioelectric effect that introduces an electric field along with antibiotics to biofilms, demonstrating significant biofilm inhibition compared to antibiotic treatment alone. Specifically, the new bioelectric effect was implemented with a superpositioned (SP) electric field of both alternating and direct current (AC and DC) and the antibiotic gentamicin (10 µg/mL). With the SP field treatment, significant biofilm reduction was demonstrated in total biomass (~ 71 %) as well as viable bacterial density (~ 400 times respected to the only antibiotic therapy) of the treated biofilms. This method was transferred to a microfluidic system using microfabricated planar electrodes. The microsystem-level implementation of the bioelectric effect also showed enhanced biofilm reduction (~ 140 % total biomass reduction improvement). The integrated system was based on the SAW sensor with the addition of coplanar thin electrodes to apply electric signals for the biofilm treatment. The chip was tested with two bacterial biofilms (Escherichia coli and Pseudomonas aeruginosa) that are clinically relevant strains. In both biofilm experiments, the integrated system demonstrated successful real-time biofilm monitoring and effective biofilm inhibition. This systematic integration of a continuous monitoring method with a novel effective treatment technique is expected to advance the state of the art in the field of managing clinical and environmental biofilms

    Optical manipulation and advanced analysis of cells using an innovative optofluidic platform

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    This doctoral research project aims to analyse complex processes of living cells using Digital Holographic Microscopy (DHM) as a three-dimensional (3D) imaging tool. DHM is a real-time, high-throughput, label-free and quantitative phase imaging technique which permits advanced cell analysis in microfluidic environment. In particular, an innovative optofluidic platform is implemented, composed of a DHM modulus and aided by holographic optical tweezers (HOT) for optical manipulation and a fluorescence modulus. This platform has been used for blood disease screening, cell manipulation studies and tracking of migrating cells. In this thesis, three main topics have been investigated. The first topic focuses on diagnostics, which plays several critical roles in healthcare. Here a novel and cost-effective approach for detecting real blood disorders such as iron-deficiency anaemia and thalassemia at lab-on-chip scale is shown. In addition, cell dynamics studies were performed by DHM. In particular, a study regarding the temporal evolution of cell morphology and volume during blue light exposure is reported. The second topic aims to investigate cell mechanics. To this end, the capabilities of HOT were used to enable the generation and the independent high-precision control of an arbitrary number of 3D optical traps. The combination of HOT and DHM provides the possibility to manipulate cells, detect nano-mechanical cell response in the pN range, and reveal cytoskeleton formation. To confirm the formation of the cytoskeleton structures after the stimulation, a fluorescence imaging system was used as control. Finally, the third topic focuses on cell manipulation using an innovative electrode-free dielectrophoretic approach (DEP) for investigating smart but simple strategies for orientation and immobilization of biological samples such as bacteria and fibroblast. In particular, the light-induced DEP is achieved using ferroelectric iron- doped lithium niobate crystal as substrate. In this way, a dynamic platform that can dynamically regulate the cell response has been developed. In this case, DHM is going to be used as a time-lapse imaging tool for the characterization of dynamic cell processes. In conclusion, the results show that DHM is a highly relevant method that allows novel insights into dynamic cell biology, with applications in cancer research and toxicity testing. In addition, this study could pave the way for detecting and quantifying circulating tumor cells and for providing multidimensional information on tumour metastasis. In this framework, the optofluidic platform is a promising tool for both identification and characterization of “foreign” cancer cells in the blood stream in order to achieve an early diagnosis
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