Cancer metastasis directly accounts for an estimated 90% of all cancer related deaths and is correlated with the presence of malignant cells in systemic circulation. This observed relationship has prompted efforts to develop a fluid biopsy, with the goal of detecting these rare cells in patient peripheral blood as surrogate markers for metastatic disease as a partial replacement or supplement to tissue biopsies. Numerous platforms have been designed, yet these have generally failed to support a reliable fluid biopsy due to poor performance parameters such as low throughput, low purity of enriched antigen positive cells, and insufficiently low detection thresholds to detect poor expressed surface markers of target cell populations. This work describes the development of a rapid cell sorting technology called Antigen Specific Lysis (ASL) based on photo-crosslinked polymer encapsulation to isolate tumor cells in suspension. In the first study, we characterize the chemical and structural properties of the surface-initiated polymer films formed directly on mammalian cell surfaces. Coated populations are shown to remain highly viable after coating formation. Biomolecular transport is examined though film coatings on cellular substrates using fluorescent, time-resolved confocal microscopy and diffusivity estimates are generated for these materials. In the next study, a lysis-based cell isolation platform is described in which marker positive cells can be specifically coated in a heterogeneous cell suspension. Anionic surfactants lyse virtually 100% of uncoated cells while fully encapsulated cells remain protected, and are then easily collected by centrifugation. We report that purified cells are released from polymeric coatings to yield viable and functional populations. We monitor cell response throughout the isolation process by multiple techniques, and report viability >80% after the sorting process. Lastly, we examine the response of process yield on the level of photoinitiator loading on target populations. Streptavidin-fluorochrome loading was quantitatively assessed on a panel of markers, both epithelial and mesenchymal, on representative model breast and lung cancer cells. We report that ASL is fundamentally capable of achieving 50-60% yield which is promising for fluid biopsy applications. Finally, both EpCAM and metastatic targeting strategies are then compared to covalently biotinylated samples to inform future robust targeting strategies.

<p>Cancer comprises a collection of diseases, all of which begin with abnormal tissue growth from various stimuli, including (but not limited to): heredity, genetic mutation, exposure to harmful substances, radiation as well as poor dieting and lack of exercise. The early detection of cancer is vital to providing life-saving, therapeutic intervention. However, current methods for detection (e.g., tissue biopsy, endoscopy and medical imaging) often suffer from low patient compliance and an elevated risk of complications in elderly patients. As such, many are looking to “liquid biopsies” for clues into presence and status of cancer due to its minimal invasiveness and ability to provide rich information about the native tumor. In such liquid biopsies, peripheral blood is drawn from patients and is screened for key biomarkers, chiefly circulating tumor cells (CTCs). Capturing, enumerating and analyzing the genetic and metabolomic characteristics of these CTCs may hold the key for guiding doctors to better understand the source of cancer at an earlier stage for more efficacious disease management.</p><p> The isolation of CTCs from whole blood, however, remains a significant challenge due to their (i) low abundance, (ii) lack of a universal surface marker and (iii) epithelial-mesenchymal transition that down-regulates common surface markers (e.g., EpCAM), reducing their likelihood of detection via positive selection assays. These factors potentiate the need for an improved cell isolation strategy that can collect CTCs via both positive and negative selection modalities as to avoid the reliance on a single marker, or set of markers, for more accurate enumeration and diagnosis.</p><p> The technologies proposed herein offer a unique set of strategies to focus, sort and template cells in three independent microfluidic modules. The first module exploits ultrasonic standing waves and a class of elastomeric particles for the rapid and discriminate sequestration of cells. This type of cell handling holds promise not only in sorting, but also in the isolation of soluble markers from biofluids. The second module contains components to focus (i.e., arrange) cells via forces from acoustic standing waves and separate cells in a high throughput fashion via free-flow magnetophoresis. The third module uses a printed array of micromagnets to capture magnetically labeled cells into well-defined compartments, enabling on-chip staining and single cell analysis. These technologies can operate in standalone formats, or can be adapted to operate with established analytical technologies, such as flow cytometry. A key advantage of these innovations is their ability to process erythrocyte-lysed blood in a rapid (and thus high throughput) fashion. They can process fluids at a variety of concentrations and flow rates, target cells with various immunophenotypes and sort cells via positive (and potentially negative) selection. These technologies are chip-based, fabricated using standard clean room equipment, towards a disposable clinical tool. With further optimization in design and performance, these technologies might aid in the early detection, and potentially treatment, of cancer and various other physical ailments.</p> / Dissertation

Les bactéries, organismes les plus abondants de notre planète, ont un rôle fondamental dans le fonctionnement des écosystèmes. En dépit de leur importance, la caractérisation des communautés bactériennes (microbiotes) demeure encore aujourd’hui très incomplète. Ceci a pour origine l’impossibilité de complètement décrypter taxonomiquement et fonctionnellement les microbiotes de ces écosystèmes et donc à appréhender la diversité bactérienne dans son ensemble que ce soit par des approches culturales (avec seulement 1% de bactéries cultivables) ou par des approches metagénomiques limitées par les biais d’extraction, de séquençage et d’analyse. Les travaux entrepris dans le cadre de cette thèse visent à développer une nouvelle voie exploratoire passant par le fractionnement des microbiotes afin d’en étudier séparément les génomes des différentes populations ou groupes de populations, leur somme devant permettre de reconstituer un metagénome complet. Cet objectif requiert le développement d’un outil pour la sélection spécifique de bactéries (sur des critères taxonomiques ou fonctionnels) et leur isolement du reste des microorganismes non ciblés. Les travaux de thèse ont porté sur le développement d’une approche de marquage magnétique des bactéries basée sur l’hybridation moléculaire (hybridation in situ) complétée par celui d’un outil de tri microfluidique. Deux méthodes ont été développées, MISH et HCR, ciblant le gène de l’ARNr 23S, chacune reposant sur la formation, lors du processus d’hybridation, d’une structure secondaire en arborescence (MISH) ou ordonnée (HCR) permettant le greffage de nanoparticules magnétiques. Les résultats obtenus illustrent le potentiel des deux approches d’abord pour le marquage spécifique de bactéries cibles (E.coli et Pseudomonas putida) en conditions de culture au laboratoire puis dans un second temps dans des échantillons de sol. Le tri microfluidique a également été optimisé par le développement d’un nouveau dispositif de tri magnétique permettant la séparation des cellules marquées sous flux continu faisant appel à l’injection d’un polymère composite magnétique pour intégrer au fond du microcanal une série de bandes parallèles magnétiques. La fonctionnalité du dispositif a été démontrée, sa simplicité de fabrication en faisant un outil de choix pour une application en routine dans les laboratoires d’écologie microbienne. En dépit de résultats prometteurs toute cette nouvelle approche d’étude de la diversité bactérienne environnementale nécessite encore de nombreuses étapes d’optimisation. / Bacteria, the most abundant organisms on our planet, have a fundamental role in ecosystem functioning. Despite their importance, the characterization of bacterial communities is today still incomplete. This is due to the impossibility of completely decomposing taxonomically and functionally the microbial communities of these ecosystems and as a consequence to apprehend the whole bacterial diversity, either by cultural approaches (with only 1% of culturable bacteria) or by metagenomic, a limited approaches cause by biases in extraction, sequencing and analysis. The work undertaken in this thesis aims to develop a new exploratory pathway through the fractionation of microbiota in order to study separately genomes of different populations or groups of populations. Their sum should enable reconstitution of complete metagenome. This objective requires the development of a tool for the specific selection of bacteria (on taxonomic or functional criteria) and their isolation from the rest of the non-target microorganisms. The thesis work focused on the development of a magnetic labeling approach for bacteria based on molecular hybridization (in situ hybridization) complete by development of a microfluidic cell-sorting tool. Two methods have been developed, MISH and HCR, targeting the 23S rRNA gene, each based on the formation, during the hybridization process, of a secondary random structure (MISH) or organized structure (HCR) enabling binding to magnetic nanoparticles. Results obtained illustrate the potential of the two approaches initially for the specific labeling of target bacteria (E.coli and Pseudomonas putida) under laboratory conditions and then in soil samples. The microfluidic sorting was also optimized by the development of a novel magnetic cell-sorting device allowing the separation of the labeled cells under continuous flow using the injection of a magnetic composite polymer to integrate a series of magnetic parallel strips at the bottom of the microchannel. The proper functioning of the sorting device has been demonstrated, its simple production making it a tool of choice for a routine application in laboratories of microbial ecology. Despite promising results all this new approach for studying environmental bacterial diversity is still requiring many optimization steps.

Diversos tipos celulares nas mais variadas condições têm sido usados como doadores de núcleo para a TN. Ainda não está claro se o estado fisiológico destas células afeta o posterior desenvolvimento dos embriões. Neste trabalho, testou-se a hipóese que fibroblastos bovinos em processo de MCP podem ser reprogramados na transferência nuclear. Fibroblastos foram cultivados até atingirem 60% de confluência, sincronizados por restrição de soro durante 24h e em seguida a MCP foi analisada por citometria de fluxo com a ténica da Anexina V/Iodeto de propídeo. Células Anexina positivas (MCP) e Anexinanegativas (Vivas) foram separadas por citometria de fluxo e utilizadas para a TNS. Céulas não coradas e não separadas no citômetro serviram como controle (Controle). Os embriões reconstruídos foram avaliados quanto à fusão, clivagem (2º dia de cultivo), blastocisto (7º dia) e prenhez (D30, D60 e nascimento). O índice de MCP dos blastocistos obtidos foi determinado. Os resultados foram analisados pela ANOVA ou teste de X2 com nível de significância de 5%. Não houve efeito nas taxas de fusão (p>0,05). Embriões reconstruídos com células MCP tiveram menor taxa de clivagem e formação de blastocistos (72,7% e 18,8%, respectivamente) em comparação ao grupo reconstruído com células Vivas (83,4% e 34,7%, respectivamente; p<0,05), não diferindo dos embriões Controle (77,3% e 27,3%, respectivamente; p>0,05). O índice de MCP do grupo de embriões MCP foi similar aos índices dos embriões clonados a partir de células Vivas e Controle (p>0,05). Após a transferência para receptoras, os grupos MCP, Vivas e Controle não diferiram com relação à taxa de prenhez aos 30d (18,1%, 13,3% e 27,5%, respectivamente; p>0,05). Entretanto aos 60d, a perda gestacional no grupo MCP (25%) foi inferior a do grupo Vivas (100%) e Controle (62,5%). Somente um nascimento, do grupo MCP (4,5% dos embriões transferidos), foi obtido no experimento. Conclui-se que células em processo de MCP, podem ser reprogramadas quando utilizadas como doadoras de núcleo na técnica de transferência nuclear, podendo estabelecer gestações e nascimentos, entretanto houve um efeito prejudicial nas taxas de desenvolvimento embrionário até o estádio de blastocisto assim como houve aumento do índice de MCP nos embriões reconstruídos. / It is not clear if the physiological status of the cells can affect further embryonic development in NT. We hypothesized that adult bovine fibroblasts in PCD can be reprogrammed when used as nuclear donors for cloning. Fibroblasts were cultivated until 60% confluency, synchronized by serum starvation for 24 h and stained with Annexin V and Propidium iodide (PI) by flow citometry. Annexin positive cells (PCD cells) and Annexin negative cells (Live cells) were sorted and used for NT. Unsorted, unstained cells were used as control (Control cells). After reconstruction, fusion, cleavage (day 2 of culture), blastocyst (day 7) and pregnancy rates (day 30, 60 and birth) were recorded. Apoptotic index of the embryos was determined by TUNEL. Data were analyzed with ANOVA and Chi-square test with 5% of significance level. There was no effect on fusion rates (p>0.05). Embryos reconstructed with PCD cells had lower cleavage and blastocyst rates (72.7 and 18.8%, respectively) compared with embryos reconstructed with Live cells (83.4 and 34.7%, respectively; p<0.05). Apoptotic index in embryos produced from cells in PCD was similar compared to embryos produced from Live and Control cells (p>0.05). Pregnancy rates were similar between cloned groups on day 30 after embryo transfer (p>0.05). However it was observed a reduced pregnancy loss in PCD group on day 60 (25%) compared with Control (62.5%) and Live (100%) groups. Only one calf, from PCD cells (4.5% of the transferred embryos), has been obtained in this experiment. In conclusion, it was showed that cells in PCD process can be reprogrammed when used as nuclear donors after NT producing even live animals. However, a negative effect on embryonic development and an increase in the apoptotic index of these embryos was observed.

The precise structure of the adult Drosophila eye results from a coordinated process of cell sorting, differentiation and selective cell death in the retinal epithelium. Mutations in the gene echinus cause supernumerary pigment cells due to insufficient cell death. This study reports the identification of echinus and the characterization of its role in Drosophila retinal development. Using a combination of deletion mapping, gene expression analysis and genomic sequencing, echinus was cloned and several alleles were sequenced. echinus encodes a ~180kDa protein containing an ubiquitin hydrolase domain at its N-terminus and a polyglutamine tract at its C-terminus. echinus is expressed in the retina during pupal development and mutants of echinus have decreased levels of apoptosis during several stages of retinal development. Defects in the cell sorting process that precedes cell death are also observed in echinus loss-of-function mutants and echinus overexpression can cause defects in ommatidial rotation and the morphology of cone cells. echinus is a positive regulator of DE-cadherin and Enabled accumulation in adherens junctions of retinal epithelial cells. Genetic interactions were observed between echinus and the genes wingless, enabled and expanded. An immunofluorescence assay in Drosophila S2 cell cultured demonstrated that Echinus localizes to intracellular vesicles that do not appear to be endocytic in nature, and the C-terminal region of Echinus was shown to be necessary for this association. A protein interaction screen using an immunoprecipitation and mass spectrometry approach identified interactions between Echinus and the vesicle coat protein Clathrin, the scaffolding protein RACK1 and the casein kinase I epsilon (Dco). Co-immunoprecipitation additionally identified an interaction between Echinus and Enabled. This work has revealed echinus to be an important regulator of cell sorting and adherens junction formation in the developing retina and has identified multiple interactions between echinus and enabled, a regulator of the actin cytoskeleton.

Biological cell manipulation and analysis is one of the most investigated applications of microfluidics. In the last decade, researchers have developed means to handle and sort cells, isolate and study single cells, assay whole and lysed cells, and transfect and electroporate in microchannels. Much of this work was motivated by the observation that many external forces and fields scale favorably in the micro-regime; this is especially the case for the electrical field. This dissertation investigates further integration of electrical forces with microfluidic devices, both channel- and droplet-based, in order to generate new, flexible and more efficient tools for studying cell biology. The first part of the dissertation (Chapter 3) explores a new dielectrophoretic particle separation method in microchannels. Current electrodeless dielectrophoretic (DEP) separation techniques utilize insulating solid obstacles in a direct current (DC) or low-frequency alternating current (AC) field, while this novel method employs an oil droplet acting as an insulating hurdle between two electrodes. Since the size of the droplet can be dynamically changed, the electric field gradient, and hence DEP force, becomes easily controllable and adjustable to various separation parameters. Very effective separation at the low field strength suggests that this method can also be applied to a separation of biological cells that are not sensitive to low electric potential. The second, larger part of the dissertation (Chapters 4 and 5) is focused on digital microfluidics (DMF), which is used to actuate nanoliter droplets of reagents and cells on a planar array of electrodes. It was demonstrated for the first time that DMF can be used as a method for cell culture and analysis. Several cell-based applications were implemented in DMF format including long-term culture, cell passaging, assaying and transfection. The data presented here suggest advanced performance of DMF techniques relative to standard macro-scale techniques. Cell analysis using DMF was found to be advantageous because of greatly reduced reagent and cell use, increased sensitivity, and the potential for multiplexing. Also, DMF technique for cell passaging demonstrated faster and more straightforward manipulation of cells than the standard techniques. In addition, no adverse effects of actuation by DMF were observed in assays for cell viability, proliferation, and biochemistry. The new DMF platform for long-term mammalian cell culture represents the first microfluidic implementation of any kind of all of the steps required for mammalian cell culture – cell seeding, growth, detachment, and re-seeding on a fresh surface. In addition, it is the first demonstration of long-term cell culture in nanoliter droplets. Cells handled in this manner exhibited growth characteristics and morphology comparable to those cultured in standard tissue culture vessels. We anticipate that the DMF cell culture and analysis techniques presented here will be useful in myriad applications that would benefit from automated mammalian cell culture.

Biological cell manipulation and analysis is one of the most investigated applications of microfluidics. In the last decade, researchers have developed means to handle and sort cells, isolate and study single cells, assay whole and lysed cells, and transfect and electroporate in microchannels. Much of this work was motivated by the observation that many external forces and fields scale favorably in the micro-regime; this is especially the case for the electrical field. This dissertation investigates further integration of electrical forces with microfluidic devices, both channel- and droplet-based, in order to generate new, flexible and more efficient tools for studying cell biology. The first part of the dissertation (Chapter 3) explores a new dielectrophoretic particle separation method in microchannels. Current electrodeless dielectrophoretic (DEP) separation techniques utilize insulating solid obstacles in a direct current (DC) or low-frequency alternating current (AC) field, while this novel method employs an oil droplet acting as an insulating hurdle between two electrodes. Since the size of the droplet can be dynamically changed, the electric field gradient, and hence DEP force, becomes easily controllable and adjustable to various separation parameters. Very effective separation at the low field strength suggests that this method can also be applied to a separation of biological cells that are not sensitive to low electric potential. The second, larger part of the dissertation (Chapters 4 and 5) is focused on digital microfluidics (DMF), which is used to actuate nanoliter droplets of reagents and cells on a planar array of electrodes. It was demonstrated for the first time that DMF can be used as a method for cell culture and analysis. Several cell-based applications were implemented in DMF format including long-term culture, cell passaging, assaying and transfection. The data presented here suggest advanced performance of DMF techniques relative to standard macro-scale techniques. Cell analysis using DMF was found to be advantageous because of greatly reduced reagent and cell use, increased sensitivity, and the potential for multiplexing. Also, DMF technique for cell passaging demonstrated faster and more straightforward manipulation of cells than the standard techniques. In addition, no adverse effects of actuation by DMF were observed in assays for cell viability, proliferation, and biochemistry. The new DMF platform for long-term mammalian cell culture represents the first microfluidic implementation of any kind of all of the steps required for mammalian cell culture – cell seeding, growth, detachment, and re-seeding on a fresh surface. In addition, it is the first demonstration of long-term cell culture in nanoliter droplets. Cells handled in this manner exhibited growth characteristics and morphology comparable to those cultured in standard tissue culture vessels. We anticipate that the DMF cell culture and analysis techniques presented here will be useful in myriad applications that would benefit from automated mammalian cell culture.

The precise structure of the adult Drosophila eye results from a coordinated process of cell sorting, differentiation and selective cell death in the retinal epithelium. Mutations in the gene echinus cause supernumerary pigment cells due to insufficient cell death. This study reports the identification of echinus and the characterization of its role in Drosophila retinal development. Using a combination of deletion mapping, gene expression analysis and genomic sequencing, echinus was cloned and several alleles were sequenced. echinus encodes a ~180kDa protein containing an ubiquitin hydrolase domain at its N-terminus and a polyglutamine tract at its C-terminus. echinus is expressed in the retina during pupal development and mutants of echinus have decreased levels of apoptosis during several stages of retinal development. Defects in the cell sorting process that precedes cell death are also observed in echinus loss-of-function mutants and echinus overexpression can cause defects in ommatidial rotation and the morphology of cone cells. echinus is a positive regulator of DE-cadherin and Enabled accumulation in adherens junctions of retinal epithelial cells. Genetic interactions were observed between echinus and the genes wingless, enabled and expanded. An immunofluorescence assay in Drosophila S2 cell cultured demonstrated that Echinus localizes to intracellular vesicles that do not appear to be endocytic in nature, and the C-terminal region of Echinus was shown to be necessary for this association. A protein interaction screen using an immunoprecipitation and mass spectrometry approach identified interactions between Echinus and the vesicle coat protein Clathrin, the scaffolding protein RACK1 and the casein kinase I epsilon (Dco). Co-immunoprecipitation additionally identified an interaction between Echinus and Enabled. This work has revealed echinus to be an important regulator of cell sorting and adherens junction formation in the developing retina and has identified multiple interactions between echinus and enabled, a regulator of the actin cytoskeleton.

In the mammalian embryo, between 3.5 and 4.5 days after fertilisation, the cells of the inner cell mass evolve from a uniform aggregate to an ordered structure with two distinct tissue layers - the primitive endoderm and epiblast. It was originally assumed that cells differentiated to form these layers in situ, but more recent evidence suggests that both cell types arise scattered throughout the inner cell mass, and it is thus proposed that the tissue layers self-organise by physical mechanisms after the specification of the two cell types. We have developed a computational model based on the subcellular element method to combine theoretical and experimental work and elucidate the mechanisms that drive this self-organisation. The subcellular element method models each cell as a cloud of infinitesimal points that interact with their nearest neighbours by local forces. Our method is built around the introduction of a tensile cortex in each cell by identifying boundary elements and using a Delaunay triangulation to define a network of forces that act within this boundary layer. Once the cortex has been established, we allow the tension in the network to vary locally at interfaces, modelling the exclusion of myosin at cell-cell interfaces and consequent reduction in tension. The model is validated by testing the simulated interfaces in cell doublets and comparing to experimental data and previous theoretical work. Furthermore, we introduce dynamic tension to model blebbing in primitive endoderm cells. We investigate the effects of cortical tension, differential interfacial tension, and blebbing on interfaces, rearrangement, and sorting. By establishing quantitative measurements of sorting we produce phase diagrams of sorting magnitude given system parameters and find that robust sorting in a 30 cell aggregate is best achieved by a combination of differential interfacial tension and blebbing.

A fixed population of oocytes within primordial follicles, established prior to or just after birth has been firmly believed to support the postnatal mammalian ovary throughout an individual’s reproductive lifespan. However, the identification and isolation of cells from adult mammalian ovaries characterised by the expression of both germ and stem cell markers, suggest the presence of mitotically active cells, termed oogonial stem cells (OSCs) that may have the potential to produce new oocytes in the postnatal mammalian ovary. Putative OSCs have been isolated from adult tissues of several mammalian species, including rodents and humans. Upon reintroduction to the ovarian niche, human and rodent OSCs have generated new oocyte like structures which, at least in rodents, have generated functional oocytes capable of fertilisation and subsequent embryonic development to produce healthy offspring. We hypothesised that OSCs could be isolated from adult bovine ovaries and upon establishment within the appropriate ovarian niche could initiate successful oogenesis. To investigate this hypothesis, we have utilised fluorescently activated cell sorting (FACS) to isolate putative bovine OSCs (bOSCs) and an ovarian aggregate model, in vitro and in vivo to explore the oogenic potential of these cells. Putative bOSCs were successfully isolated by FACS based on the cell surface expression of germ cell marker DDX4 and established in culture. Pluripotency (LIN28 and OCT4) and germ (IFITM3, PRDM1, C-KIT and DAZL) cell associated genes were expressed in putative bOSCs established in culture. However, DDX4 transcripts were not consistently observed throughout bOSC culture. Aggregation of putative bOSCs with neonatal murine ovarian somatic cells to form chimeric ovarian aggregates, cultured in a hanging drop model for 7 days maintained germ cell phenotype, marked by DAZL expression. A subpopulation of putative bOSCs showed a spherical morphology, an increase in cell size and an association with neighbouring cells. Xenotransplantation of chimeric ovarian aggregates to the kidney capsule of immune deficient mice for 21 days generated multi-laminar follicles and structures with morphological similarities to primordial follicles (termed pre-primordial follicle-like structures). RNA Scope was unsuccessful in determining the origin of oocytes within chimeric ovarian aggregates. However, oocytes from pre-antral follicles in chimeric ovarian aggregates (n=6; 60.9± 3.6 μm, mean ± SEM) were significantly (P < 0.0001) larger than murine oocytes (n=38; 34.5± 1 μm, mean ± SEM) aggregated with murine ovarian somatic cells as positive controls, suggesting that these oocytes are undergoing different growth dynamics. This work has shown that putative bOSCs characterised by the expression of pluripotency and germ cell associated genes are present within adult bovine ovarian tissue and can be isolated using FACS and established in culture. These data also suggest that putative bOSCs may have the potential to undergo oogenesis and illustrate the potential use of these cells as a tool to investigate germ cell differentiation.