Cell Microarray Technologies for High-Throughput Cell-Based Biosensors

Due to the recent demand for high-throughput cellular assays, a lot of efforts have been made on miniaturization of cell-based biosensors by preparing cell microarrays. Various microfabrication technologies have been used to generate cell microarrays, where cells of different phenotypes are immobilized either on a flat substrate (positional array) or on particles (solution or suspension array) to achieve multiplexed and high-throughput cell-based biosensing. After introducing the fabrication methods for preparation of the positional and suspension cell microarrays, this review discusses the applications of the cell microarray including toxicology, drug discovery and detection of toxic agents.ope

High-throughput processing and analysis of marine-based biomaterials and cells

Tese de Doutoramento em Engenharia BiomédicaAs the field of Tissue Engineering (TE) progresses, new technology are essential to accelerate the identification of potentially translatable approaches for the repair of tissues damaged due to disease or trauma. The development of high-throughput and combinatorial technologies is helping to speed up research that is applicable to all aspects of the TE paradigm. In this thesis, interactions of cells with many diverse materials in both two- and three-dimensions was assessed rapidly through the use of superhydrophobic (SH) chips for rapid outcome measurements of cell-material or cell-cell combinations. The main hypothesis is that flat biomimetic SH surfaces patterned with transparent superhydrophilic (SL) spots were used to individual pattern biomaterials with precise shape and pre-determined height, by controlling the volume dispensed in each wettable spot. Initially, distinct combinations of nanostructured films were produced using layer-by-layer methodology and their morphological, physicochemical, and biological properties were analyzed using glass slides and then validated on-chip. Inspired by the composition of the adhesive proteins in mussels, thin films containing dopamine-modified hyaluronic acid were studied. The flat configuration of the SH chip allowed to perform a series of nondestructive and non-conventional measurements directly on the individual spots. In situ adhesion properties were directly measured in each wettable spot, showing that nanostructured films richer in dopamine promote the adhesion compared to control films (hyaluronic acid and alginate films – two polysaccharides often regarded as good natural adhesives – were assembled in parallel). In vitro tests showed an enhanced cell adhesion for the films with more catechol groups. Combining two biomimetic concepts we developed devices with multifunctional capabilities. One approach was based on two-sided film made almost entirely from polystyrene onto which the properties of both lotus leaves and mussel adhesive were incorporated. On one side of the film, imparting micro and nanometer scale hierarchical roughness yields superhydrophobicity and water repellency, providing rapid fluid flow and the basis for microfluidic devices, as such synthetic blood or fluid flow vessels. On second approach, SH microarray based on the so-called lotus effect were produced, onto which arrays of micro-indentations allow the fixing of liquid droplets, based on the rose-petal effect. Such platforms were able to sustain arrays of quasi-spherical microdroplets, allowing the isolation and confinement of different combinations of substances and living cells. Distinct compartmentalized physical, chemical, and biological processes were monitored individually in each droplet. In addition, taking the advantage of controlled positional adhesion and minimum contact with a solid substrate, we developed a novel hanging spherical drop system for anchoring arrays of droplets of cell suspension. By facing the chip downward it was possible to generate independent spheroids bodies in a high throughput manner, in order to mimic in vivo tumor models in a lab-on-chip scale. The system was validated for drug screening purposes and the toxicity of the anti-cancer drug doxorubicin in cell spheroids was tested and compared to monolayer cells culture. Finally, high-throughput fabrication of alginate hydrogel particles of specific sizes and shapes was developed using a droplet microarray. The method was based on the formation of arrays of droplets of pre-hydrogel solutions on SH-SL patterns using the process of discontinuous de-wetting, followed by their gelation via the parallel addition of the crosslinker to the individual droplets via the sandwiching method. The viability of living cells incorporated within the hydrogel particles was evaluated showing to be higher during the long-term cultivation than in the case of cells cultured in the bulk three-dimensional hydrogel matrix. In conclusion, SH platforms patterned with wettable spots used in this thesis proved to be compatible with a complete study of both two- and three-dimensional biomaterial-cell interactions, comprising a wide set of factors as biomaterials characterization and in vitro testing. Although much of the work performed is only applicable for in vitro studies, future methods may translate into rapid screening of these approaches in vivo.Com o progresso da área de engenharia de tecidos (ET), novas tecnologias são essenciais para acelerar a identificação de abordagens potencialmente adaptáveis para a reparação de tecidos danificados devido a doenças ou traumatismo. O desenvolvimento de testes expeditos e tecnologias combinatórias está a ajudar a acelerar a investigação que é aplicável para todos os exemplos dos paradigmas de ET. Nesta tese, as interações das células com vários materiais, tanto em duas como em três dimensões, foram rapidamente analisadas através de chips superhidrofóbicos (SH), de modo a obter medições rápidas das combinações entre células-materiais ou células-células. A hipótese principal é que superfícies planas SH padronizadas com regiões superhidrofílicas (SL) transparentes foram usadas para depositar biomateriais com forma e altura precisas, controlando o volume depositado em cada área molhável. Inicialmente, combinações distintas de filmes nano-estruturados foram produzidos através da metodologia de camada-sobre-camada, e as suas propriedades morfológicas, físico-químicas e biológicas foram analisadas em lâminas de vidro, e posteriormente, validadas em chip, tendo como inspiração a composição das proteínas adesivas existente nos mexilhões. Filmes finos compostos por ácido hialurónico modificado com dopamina foram alvo de estudo. A configuração plana do chip SH permitiu executar uma série de medições não-destrutivas e não-convencionais diretamente sobre as regiões molháveis. As propriedades de adesão in situ foram diretamente medidas em cada região hidrofílica, mostrando que os filmes nano-estruturados com mais dopamina promovem a adesão, em comparação com filmes usados como controlo (filmes de ácido hialurónico e de alginato - dois polissacarídeos considerados como bons adesivos naturais - foram construídos em paralelo). Os testes in vitro mostraram uma melhor adesão celular para os filmes com mais grupos catechol. A combinação de dois conceitos bio-miméticos permitiu desenvolver dispositivos com capacidades multifuncionais. Uma primeira abordagem foi baseada num filme contendo duas faces, feito inteiramente a partir de poliestireno, integrando as tanto as propriedades das folhas de lótus as adesivas do mexilhão. Um lado do filme obtém superhidrofobicidade e alta repelência à água devido à rugosidade hierárquica e à escala micro e nano-métrica, proporcionando um fluxo de fluido rápido e uma base para novos dispositivos para microfluídica, como tal sangue sintético ou fluxo de fluidos nos canais. Na segunda abordagem, as superfícies SH baseadas no efeito de lótus foram padronizadas com micro-indentações para fixar gotas líquidas, mimetizando o efeito das pétalas de rosas. Estas plataformas foram capazes de suportar matrizes de micro-gotas quase esféricas, permitindo o isolamento e aderência de diferentes combinações de substâncias e células vivas. Diversos processos, tais como, físicos, químicos e biológicos foram monitorizados individualmente em cada gota. Além disso, tendo em conta a vantagem da adesão controlada da posição e contacto mínimo da gota com a superfície sólida, desenvolvemos um novo sistema de gota esférica pendurada de modo a fixar matrizes de gotas contendo suspensão de células. Voltando a superfície sólida para baixo, foi possível gerar corpos independentes de esferoides de uma forma expedita, de modo a mimetizar modelos in vivo, a uma escala lab-on-chip. O sistema foi validado para fins de rastreio de fármacos e a toxicidade da doxorubicina nos esferoides foi testada e comparada com a cultura de células em monocamada. Finalmente, o fabrico expedito de hidrogeis de alginato com o tamanho e forma especifica foi desenvolvido usando superfícies padronizadas de gotas. O método foi baseado na formação de matrizes de gotas de soluções pré-hidrogel nas superfícies padronizadas SH com regiões SL usando um processo molhável descontinuo. O processo de formação de gel foi obtido através da adição paralela de um agente reticulante a cada gota individualmente, usando um método “sandwich”. A viabilidade das células incorporadas dentro do hidrogel foi avaliada para um longo tempo de cultura mostrando ser mais elevada em comparação com as células incorporadas dentro de uma matriz 3D de hidrogel em massa. Em conclusão, as superfícies padronizadas SH com regiões molháveis provaram ser compatíveis com um estudo completo de interações 2D e 3D entre células e biomateriais, compreendendo um vasto conjunto de factores como a caracterização de biomateriais e testes in vitro. Embora a maioria do trabalho realizado só tem aplicabilidade para estudos in vitro, métodos futuros podem transpor para uma seleção rápida destas abordagens in vivo

Multifunctional composite hydrogels for bacterial capture, growth/elimination, and sensing applications

Hydrogels are cross-linked networks of hydrophilic polymer chains with a three-dimensional structure. Owing to their unique features, the application of hydrogels for bacterial/antibacterial studies and bacterial infection management has grown in importance in recent years. This trend is likely to continue due to the rise in bacterial infections and antimicrobial resistance. By exploiting their physicochemical characteristics and inherent nature, hydrogels have been developed to achieve bacterial capture and detection, bacterial growth or elimination, antibiotic delivery, or bacterial sensing. Traditionally, the development of hydrogels for bacterial/antibacterial studies has focused on achieving a single function such as antibiotic delivery, antibacterial activity, bacterial growth, or bacterial detection. However, recent studies demonstrate the fabrication of multifunctional hydrogels, where a single hydrogel is capable of performing more than one bacterial/antibacterial function, or composite hydrogels consisting of a number of single functionalized hydrogels, which exhibit bacterial/antibacterial function synergistically. In this review, we first highlight the hydrogel features critical for bacterial studies and infection management. Then, we specifically address unique hydrogel properties, their surface/network functionalization, and their mode of action for bacterial capture, adhesion/growth, antibacterial activity, and bacterial sensing, respectively. Finally, we provide insights into different strategies for developing multifunctional hydrogels and how such systems can help tackle, manage, and understand bacterial infections and antimicrobial resistance. We also note that the strategies highlighted in this review can be adapted to other cell types and are therefore likely to find applications beyond the field of microbiology

Microdevices and Microsystems for Cell Manipulation

Microfabricated devices and systems capable of micromanipulation are well-suited for the manipulation of cells. These technologies are capable of a variety of functions, including cell trapping, cell sorting, cell culturing, and cell surgery, often at single-cell or sub-cellular resolution. These functionalities are achieved through a variety of mechanisms, including mechanical, electrical, magnetic, optical, and thermal forces. The operations that these microdevices and microsystems enable are relevant to many areas of biomedical research, including tissue engineering, cellular therapeutics, drug discovery, and diagnostics. This Special Issue will highlight recent advances in the field of cellular manipulation. Technologies capable of parallel single-cell manipulation are of special interest

Hydrogel encapsulated droplet interface bilayer networks as a chassis for artificial cells and a platform for membrane studies

There has been increasing interest in droplet interface bilayers (DIBs) as novel devices for the study of lipid membranes and the development of artificial cell systems. Although DIBs have demonstrated to be useful in a number of laboratory applications, their wider use is hampered by a limited ability to exist untethered and remain mechanically stable beyond controlled laboratory environments. In this thesis, a microfluidic system is developed which enables the facile generation of hydrogel-encapsulated DIB networks which are freestanding and can exist in air, water and oil environments, without compromise to their ability to interface with the surrounding environment. Electrophysiology is employed in order to demonstrate the formation of bilayers between the encapsulated DIBs (eDIBs) and their external environment, achieved via the incorporation of the transmembrane pore α-Hemolysin. The eDIBs produced here are able to form higher-order structures akin to tissues via their assembly and adherence to one another, further demonstrating their potential to act as a chassis for artificial cells. Furthermore, the potential of eDIBs to be used as a platform for membrane studies is demonstrated via their use as a high-throughput array for membrane disruption fluorescence measurements using a plate reader, which makes use of the ability of eDIBs to be generated in large numbers as well as to be mechanically handled and placed in the wells of a 96-well plate. Fluorescence measurements were taken on up to 47 eDIBs simultaneously, and were able to detect bilayer leakage through pores as well as bilayer failure. The above experiments comprise the design, manufacture and use of a novel kind of DIB construct as a chassis for artificial cells and a platform for high-throughput membrane studies. It is proposed that eDIBs may help in realising the unfulfilled potential of DIB networks in applications in healthcare and beyond

Self-powered mobile sensor for in-pipe potable water quality monitoring

Traditional stationary sensors for potable-water quality monitoring in a wireless sensor network format allow for continuous data collection and transfer. These stationary sensors have played a key role in reporting contamination events in order to secure public health. We are developing a self-powered mobile sensor that can move with the water flow, allowing real-time detection of contamination in water distribution pipes, with a higher temporal resolution. Functionality of the mobile sensor was tested for detecting and monitoring pH, Ca2+, Mg2+, HCO3-/CO32-, NH4+, and Clions. Moreover, energy harvest and wireless data transmission capabilities are being designed for the mobile sensor

Advancing the cell culture landscape:the instructive potential of artificial and natural geometries

This research focuses on how surface structures can influence the behaviour of cells. There is a great diversity of surface structures, which makes the identification of an optimal physical environment for a specific phenotype difficult. Therefore, platforms that allow screening of many different designs at the same time facilitate the identification of an optimal cultural environment. Using the TopoChip, which contains 2176 unique microtopographies, structures have been identified that support the tenocyte phenotype, the primary cell type of the tendon. In addition, this also applies to mesenchymal stem cells (MSCs), which experience an activation of tendon-related genes. Furthermore, the library has been creatively expanded by using natural surface topographies that cause unique cell behaviour, such as promoting osteogenesis

Puce à cellules multiplexée pour l'étude de réponses cellulaires parallélisées

The work reported in this thesis focuses on the development of a multiplexed cell chip for the study of parallelized cellular responses. The lineage of cells from Prostate cancer LNCaP cells, were used as a study model thanks to their ability to secrete prostate-specific antigen (PSA) and β-2-microglobulin (B2M) in response to induction by hormones such as dihydrotestosterone (DHT). We were able to detect in real time these label-free molecules and their secretion by small populations of adherent LNCaP cells (from 1 to 100 cells) at specified positions on a SPRi biochip. Three different approaches were considered for this biochip. The first was to pattern the gold surface of a SPRi slide to obtain microwells whose bottom reveals the gold surface (cytophilic area) and an outer shell composed of polystyrene (cytophobe) to create an adhesive/non-adhesive surface for cell culture. Antibodies were immobilized in a controlled manner in the microwells using a piezo electric spotter. In this miniaturized system, different cell lines were co-cultured on a surface of 1 cm², paving the way for multiplexing. A small population of cells (1 to 100) was deposited in an automated manner into each microwell. In order to maintain the cells in a hydrated environment during deposition, a biocompatible alginate polymer was used. This method allows the encapsulation of cells in a very small volume (<50 nL). The ability of the hydrogel to maintain the encapsulated cells in a given position on the support led to the design of a second approach for the production of the biochip. In this second approach the surface is not altered and biological compounds (antibodies and cells) are directly deposited in an automated manner on the gold layer. Finally, a last approach was developed by immobilizing the cells on a patterned substrate placed in front of the sensitive layer SPRi. In all three approaches, the kinetics of PSA secretion and secreted B2M could be followed by SPRiLes travaux présentés dans cette thèse concernent le développement d'une puce à cellules multiplexée pour l'étude de réponses cellulaires parallélisées. La lignée de cellules issues de cancer de la prostate, les cellules LNCaP, ont servi de modèle d'étude grâce à leur capacité à sécréter l'antigène prostate-spécifique (PSA) et la β-2-microglobuline (B2M) en réponse à l'induction par des hormones telles que la dihydrotestostérone (DHT). Nous avons ainsi pu détecter en temps réel et sans marquage ces molécules lors de leur sécrétion par de petites populations de cellules LNCaP adhérentes (de 1 à 100 cellules) à des positions déterminées sur une biopuce SPRi. Trois approches différentes ont été envisagées pour cette biopuce. La première consistait à microtexturer la surface d'or d'un support de SPRi afin d'obtenir des micropuits dont le fond révèle la surface d'or (région cytophile) et dont l'extérieur est composé de polystyrène (cytophobe) afin de créer un contraste d'adhésion pour la culture cellulaire. Des anticorps ont pu être immobilisés de façon contrôlée dans les micropuits grâce à un automate de micro-dépôt non contact. Dans ce système miniaturisé, différentes lignées cellulaires ont pu être co-cultivées sur une surface de 1 cm², ouvrant la voie au multiplexage. Une petite population de cellules (de 1 à 100) a été déposée de façon automatisée dans chaque micropuits. Afin de maintenir les cellules dans un milieu hydraté au cours du dépôt, un polymère d'alginate biocompatible a été utilisé. Cette méthode permet l'encapsulation de cellules dans un très petit volume (< 50 nL). La capacité de cet hydrogel à maintenir les cellules encapsulées à une position donnée sur le support a conduit à la conception d'une deuxième approche pour la fabrication de la biopuce. En effet dans cette approche la surface n'est pas modifiée et les composés biologiques (anticorps et cellules) sont directement déposés de façon automatisée sur la couche d'or. Enfin une dernière approche a été développée en immobilisant cette fois-ci les cellules sur un support microtexturé placé en face de la couche sensible de SPRi. Dans les trois approches, les cinétiques de sécrétion du PSA et de la B2M sécrétées ont pu être suivies par SPR

Industrial lab-on-a-chip: design, applications and scale-up for drug discovery and delivery

Microfluidics is an emerging and promising interdisciplinary technology which offers powerful platforms for precise production of novel functional materials (e.g., emulsion droplets, microcapsules, and nanoparticles as drug delivery vehicles- and drug molecules) as well as high-throughput analyses (e.g., bioassays, detection, and diagnostics). In particular, multiphase microfluidics is a rapidly growing technology and has beneficial applications in various fields including biomedicals, chemicals, and foods. In this review, we first describe the fundamentals and latest developments in multiphase microfluidics for producing biocompatible materials that are precisely controlled in size, shape, internal morphology and composition. We next describe some microfluidic applications that synthesize drug molecules, handle biological substances and biological units, and imitate biological organs. We also highlight and discuss design, applications and scale up of droplet- and flow-based microfluidic devices used for drug discovery and delivery. © 2013 Elsevier B.V. All rights reserved