26 research outputs found
Techniques to stimulate and interrogate cell–cell adhesion mechanics
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
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
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
Developing new 3D hydrogel models of the human mammary gland to investigate breast cancer initiation
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Genetic Analysis and Cell Manipulation on Microfluidic Surfaces
Personalized cancer medicine is a cancer care paradigm in which diagnostic and therapeutic strategies are customized for individual patients. Microsystems that are created by Micro-Electro-Mechanical Systems (MEMS) technology and integrate various diagnostic and therapeutic methods on a single chip hold great potential to enable personalized cancer medicine. Toward ultimate realization of such microsystems, this thesis focuses on developing critical functional building blocks that perform genetic variation identification (single-nucleotide polymorphism (SNP) genotyping) and specific, efficient and flexible cell manipulation on microfluidic surfaces. For the identification of genetic variations, we first present a bead-based approach to detect single-base mutations by performing single-base extension (SBE) of SNP specific primers on solid surfaces. Successful genotyping of the SNP on exon 1 of HBB gene demonstrates the potential of the device for simple, rapid, and accurate detection of SNPs. In addition, a multi-step solution-based approach, which integrates SBE with mass-tagged dideoxynucleotides and solid-phase purification of extension products, is also presented. Rapid, accurate and simultaneous detection of 4 loci on a synthetic template demonstrates the capability of multiplex genotyping with reduced consumption of samples and reagents. For cell manipulation, we first present a microfluidic device for cell purification with surface-immobilized aptamers, exploiting the strong temperature dependence of the affinity binding between aptamers and cells. Further, we demonstrate the feasibility of using aptamers to specifically separate target cells from a heterogeneous solution and employing environmental changes to retrieve purified cells. Moreover, spatially specific capture and selective temperature-mediated release of cells on design-specified areas is presented, which demonstrates the ability to establish cell arrays on pre-defined regions and to collect only specifically selected cell groups for downstream analysis. We also investigate tunable microfluidic trapping of cells by exploiting the large compliance of elastomers to create an array of cell-trapping microstructures, whose dimensions can be mechanically modulated by inducing uniform strain via the application of external force. Cell trapping under different strain modulations has been studied, and capture of a predetermined number of cells, from single cells to multiple cells, has been achieved. In addition, to address the lack of aptamers for targets of interest, which is a major hindrance to aptamer-based cell manipulation, we present a microfluidic device for synthetically isolating cell-targeting aptamers from a randomized single-strand DNA (ssDNA) library, integrating cell culturing with affinity selection and amplification of cell-binding ssDNA. Multi-round aptamer isolation on a single chip has also been realized by using pressure-driven flow. Finally, some perspectives on future work are presented, and strategies and notable issues are discussed for further development of MEMS/microfluidics-based devices for personalized cancer medicine
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 /
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Experimentation and Multiphysical Modeling of Bioanalytical Microdevices
Bioanalytics involves quantitative measurements of complex biological samples that contain metabolites, DNA, RNA, and proteins. Efficient sample preparation for downstream analysis and sensitive detection of analytes can be achieved via bioanalytical microdevices. Fully realizing the potential of these devices requires tool characterization and bioprocess optimization, in addition to understanding device physics. Therefore, this thesis introduces multiphysical modeling and experimentation of microdevices, with applications to diabetes care and single-cell analysis.
To understand the physics of viscometric glucose microsensors, this thesis presents a model of the sensor, which couples the fluid flow with vibrating diaphragms. The model is used to predict the sensor response to glucose via theory of squeeze-film damping and vibrations of pre-stressed plate. A first-principle-based model resulting from the theory can be evaluated from the device's geometric and material properties, and quantitatively determines the device response to vibrational excitations at varying glucose concentrations.
Next, this thesis introduces a theoretical model for viscometric glucose microsensors that employ harmonic microcantilever oscillation in the sensing liquid. The presented model associates the unsteady Stokes equation with the motion of a bounded viscous liquid to understand the hydrodynamic impact on the cantilever. With a proper consideration of the viscosity and bounded geometry of liquid media, the model relaxes the thin-film assumption required for the diaphragm-based model, enabling an accurate representation of fluid-structure interactions based on fundamental structural vibration and fluid flow equations.
Next, this thesis presents an experimental exploration of a hydrogel-based affinity microsensor for glucose monitoring via dielectric measurements. The microsensor incorporates a synthetic hydrogel that is attached to the device surface via in situ polymerization, which eliminates mechanical moving parts required in the viscometric glucose sensors. Changes in the dielectric properties of the hydrogel when binding reversibly with glucose molecules have been measured using a MEMS capacitive transducer to determine the glucose concentration. Experimental results demonstrate that in a glucose concentration range of 0–500 mg/dL and with a resolution of 0.35 mg/dL or better, the microsensor exhibits a repeatable and reversible response, and can potentially be useful for continuous glucose monitoring in diabetes care.
Additionally, this thesis presents a microfluidic preprocessing method that integrates single-cell picking, lysing, reverse transcription and digital polymerase chain reaction to enable the isolation, tracking and gene expression analysis at single-cell level for individual cells. The approach utilizes a photocleavable bead-based microfluidic device to synthesize and deliver stable complementary DNA for downstream gene expression analysis, thereby allowing chip-based integration of multiple reactions and facilitating the minimization of sample loss or contamination.
Finally, this thesis ends with concluding remarks and directions of future work towards continuous glucose monitoring and high-throughput single-cell genetic analysis
AN INTEGRATED MICROSYSTEM FOR BACTERIAL BIOFILM DETECTION AND TREATMENT
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
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