Electro-Mechanical Manipulation of Mammalian Cells in Suspension

Le but principal de cette thèse était de décrire le développement d'une plate-forme microfabriquée et son application pour la caractérisation mécanique des cellules mammifères vivantes. La technique emploie les forces de polarisation électriques pour emprisonner et étirer des cellules dans des champs électriques non-uniformes et variables dans le temps. Ce travail a été motivé par la sous-utilisation apparente des champs électriques pour la caractérisation mécanique des cellules; les méthodes décrites ici ont permis la caractérisation mécanique de cellules mammifères diverses et précédemment non étudiées. La caractérisation mécanique de cellules vivantes est habituellement réalisée en sondant des structures locales près de la surface des cellules. Très peu de ces techniques appliquent des contraintes uniaxiales aux cellules entières et la comparaison des données de diverses techniques est donc difficile. Par contre, des champs électriques peuvent être employés pour exercer ces forces uniaxiales sur les cellules suspendues. La plupart des cellules mammifères adoptent une géométrie quasi-sphérique lorsque suspendues dans un milieu (aqueux) liquide; ceci simplifie à la fois la manipulation des cellules ainsi que l'interprétation des données mécaniques. Les champs électriques exercent des forces sans contact mécanique significatif entre les cellules et les structures du dispositif et peuvent donc être décrits comme un « rayon tracteur », qui peut déplacer, emprisonner, ou déformer électriquement les objets polarisables telles les cellules biologiques. En plus, les électrodes nécessaires sont facilement incorporées dans les dispositifs microfabriqués, ce qui suggère que les techniques basées sur des champs électriques seront de plus en plus utilisées. La caractérisation mécanique des cellules par « électro-déformation » (ED) se place dans le contexte plus grand de l‟électro-manipulation de cellules. Afin de mieux comprendre le comportement des cellules dans les champs électriques, nous avons commencé nos études en utilisant la diélectrophorèse (DEP) pour placer des monocytes humains (U937) dans un champ électrique non-uniforme, avant d‟effectuer l'électroporation (EP) permettant la livraison de transgènes. Un ensemble d'électrodes planaires inertes microfabriquées sur un substrat de verre a été utilisé pour la DEP et l‟EP. Les propriétés diélectriques des cellules ont été estimées et la modélisation (par éléments finis) des champs électriques nous à permis de prévoir le positionnement des cellules. Le point à partir du quel les impulsions électriques ont augmenté la perméabilité des membranes cellulaires aux molécules fluorescentes et aux plasmides d'ADN----------Abstract The purpose of this study has been to describe the development and demonstration of a microfabricated platform for mechanical characterization of individual living mammalian cells in suspension. The technique uses electrical polarization forces to trap and stretch cells in time-varying, non-uniform fringing electric fields. This work was motivated by the apparent under-utilization of electrical stresses for the mechanical characterization of live cells, and the methods described here permitted mechanical characterization of diverse (previously uncharacterized) mammalian cell-types. Mechanical characterization of cells is usually achieved by probing local structures near the cell-surface, and very few techniques apply uniaxial stresses to whole individual cells. Most mammalian cells adopt a relatively simple (spherical) geometry when they are suspended in a liquid (aqueous) medium. This simplifies cell-manipulation and permits relatively straight-forward interpretation of mechanical data. Mammalian cells are increasingly being used outside of their natural environments, for example within microfluidic devices, which requires precise cell-manipulation protocols. Present miniaturization trends within experimental biotechnology are producing new tools for the precise manipulation of individual living cells, and electric fields feature prominently within this context. Electric fields exert forces on cells without the requirement of mechanical contact between cells and device structures and can therefore be described as “tractor beams”, which can move, trap, or deform electrically polarisable objects such as biological cells. Mechanical characterization of cells by electro-deformation (ED) will be described within the larger context of cell electro-manipulations. To better understand the behaviour of cells in electric fields, we used dielectrophoresis (DEP) to position human monocytes (U937) within a non-uniform electric field prior to electro-poration (EP) for gene delivery. DEP positioning and EP pulsing were both accomplished using a common set of inert planar electrodes, micro-fabricated on a glass substrate. A single-shell model of the cell‟s dielectric properties and finite-element modeling of the electric field distribution permitted us to predict the major features of experimentally observed cell positioning. The extent to which electric pulses increased the permeability of the cell-membranes to florescent molecules and to pEGFP-Luc DNA plasmids were found to depend on prior positioning. For a given set of pulse parameters, EP was either irreversible (resulting in cytolysis), reversible (leading to gene delivery), or not detectable

Vortex Characterization for Engineering Applications

Realistic engineering simulation data often have features that are not optimally resolved due to practical limitations on mesh resolution. To be useful to application engineers, vortex characterization techniques must be sufficiently robust to handle realistic data with complex vortex topologies. In this paper, we present enhancements to the vortex topology identification component of an existing vortex characterization algorithm. The modified techniques are demonstrated by application to three realistic data sets that illustrate the strengths and weaknesses of our approach

Perturbation of adhesion molecule-mediated chondrocyte-matrix interactions by 4-hydroxynonenal binding: implication in osteoarthritis pathogenesis

ABSTRACT: INTRODUCTION: Objectives were to investigate whether interactions between human osteoarthritic chondrocytes and 4-hydroxynonenal (HNE)-modified type II collagen (Col II) affect cell phenotype and functions and to determine the protective role of carnosine (CAR) treatment in preventing these effects. METHODS: Human Col II was treated with HNE at different molar ratios (MR) (1:20 to 1:200; Col II:HNE). Articular chondrocytes were seeded in HNE/Col II adduct-coated plates and incubated for 48 hours. Cell morphology was studied by phase-contrast and confocal microscopy. Adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and alpha1beta1 integrin at protein and mRNA levels were quantified by Western blotting, flow cytometry and real-time reverse transcription-polymerase chain reaction. Cell death, caspases activity, prostaglandin E2 (PGE2), metalloproteinase-13 (MMP-13), mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-kappaB) were assessed by commercial kits. Col II, cyclooxygenase-2 (COX-2), MAPK, NF-kappaB-p65 levels were analyzed by Western blotting. The formation of alpha1beta1 integrin-focal adhesion kinase (FAK) complex was revealed by immunoprecipitation. RESULTS: Col II modification by HNE at MR approximately 1:20, strongly induced ICAM-1, alpha1beta1 integrin and MMP-13 expression as well as extracellular signal-regulated kinases 1 and 2 (ERK1/2) and NF-kappaB-p65 phosphorylation without impacting cell adhesion and viability or Col II expression. However, Col II modification with HNE at MR approximately 1:200, altered chondrocyte adhesion by evoking cell death and caspase-3 activity. It inhibited alpha1beta1 integrin and Col II expression as well as ERK1/2 and NF-kappaB-p65 phosphorylation, but, in contrast, markedly elicited PGE2 release, COX-2 expression and p38 MAPK phosphorylation. Immunoprecipitation assay revealed the involvement of FAK in cell-matrix interactions through the formation of alpha1beta1 integrin-FAK complex. Moreover, the modification of Col II by HNE at a 1:20 or approximately 1:200 MR affects parameters of the cell shape. All these effects were prevented by CAR, an HNE-trapping drug. CONCLUSIONS: Our novel findings indicate that HNE-binding to Col II results in multiple abnormalities of chondrocyte phenotype and function, suggesting its contribution in osteoarthritis development. CAR was shown to be an efficient HNE-snaring agent capable of counteracting these outcomes

Modern meat: the next generation of meat from cells

Modern Meat is the first textbook on cultivated meat, with contributions from over 100 experts within the cultivated meat community. The Sections of Modern Meat comprise 5 broad categories of cultivated meat: Context, Impact, Science, Society, and World. The 19 chapters of Modern Meat, spread across these 5 sections, provide detailed entries on cultivated meat. They extensively tour a range of topics including the impact of cultivated meat on humans and animals, the bioprocess of cultivated meat production, how cultivated meat may become a food option in Space and on Mars, and how cultivated meat may impact the economy, culture, and tradition of Asia