Continuous-flow separation of cells in a lab-on-a-chip using "liquid electrodes" and multiple-frequency dielectrophoresis

This thesis reports on the integration of continuous-flow cell separation method for lab-on-a-chip applications. Cell separation methods are widely used in biology to prepare samples prior to analysis. There is a need for a highly sensitive separation method that is capable to quickly isolate a particular cell type in a single manipulation step. We attempt to provide such a separation method that discriminates between cell types according to their dielectric properties such as the membrane permittivity and the cytoplasm conductivity. The dielectric properties are intrinsic to each cell type and thus prevent the need of a specific cell labeling as discriminating factor. To guaranty a high throughput, the cell separation method we propose is performed in a continuous flow. The continuous-flow cell separation method presented in this thesis makes use of electrical forces to achieve the separation of different cell types. Dielectrophoresis is a phenomenon that describes the electrical force exerted on a dielectric particle such as a biological cell in presence of a non-uniform AC electric field. The combination of several dielectrophoretic forces at multiple frequencies produces a distinct dielectric response for each cell type. The method is integrated into a microfluidic platform of 20 mm long by 15 mm wide. The microfabrication of the device consists of two successive steps of photolithography to define the metal electrodes in platinum and the microfluidic network in SU-8 photoresist. In the so-called separation chamber, an array of "liquid electrodes" is localized along the 20 µm deep central channel. The technological development of "liquid electrodes" allow us to produce horizontal dielectrophoretic force and the array of these electrodes opposes two fields of such forces. This opposition of two force fields defines an equilibrium position towards which the cells that flow through the central channel are focused. There is a relationship between the equilibrium position and the dielectric properties of the cells which allows a flow-through dielectric characterization of the cells by the cell position readout. Using this microfluidic platform that integrate a method of continuous-flow cell separation based on multiple-frequency dielectrophoresis, we succeeded in purifying fractions of viable and nonviable yeast cells that were initially mixed. Due to its sensitivity, the method also allowed to increase the infection rate of a cell culture up to 50% of parasitemia percentage, which facilitates the study of the parasite cycle. The method was finally applied to the biological issue of cell synchronization. By isolating cells that are at a particular phase within their cell cycle, our method prevents the use of metabolic agents in order to arrest a cell culture by disrupting the cell physiology. The synchronization method we proposed and based on multiplefrequency dielectrophoresis is to our best knowledge the most powerful one in terms of synchrony level reported so far

Label-free detection of Babesia bovis infected red blood cells using impedance spectroscopy on microfabricated flow cytometer

Impedance spectroscopy is a powerful tool for label-free analysis and characterisation of living cells. In this work, we achieved the detection of Babesia bovis infected red blood cells using impedance spectroscopy on a microfabricated flow cytometer. The cellular modifications caused by the intracellular parasite result in a shift in impedance which can be measured dielectrically. Thus, a rapid cell-by-cell detection with microliter amounts of reagents is possible. Unlike other diagnostic tests, this method does not depend on extensive sample pre-treatment or expensive chemicals and equipment

Tracking and synchronization of the yeast cell cycle using dielectrophoretic opacity

Cell cycle synchronization is an important tool for the study of the cell division stages and signalling. It provides homogeneous cell cultures that are of importance to develop and improve processes such as protein synthesis and drug screening. The main approach today is the use of metabolic agents that block the cell cycle at a particular phase and accumulate cells at this phase, disturbing the cell physiology. We provide here a non-invasive and label-free continuous cell sorting technique to analyze and synchronize yeast cell division. By balancing opposing dielectrophoretic forces at multiple frequencies, we maximize sensitivity to the characteristic shape and internal structure changes occurring during the yeast cell cycle, allowing us to synchronize the culture in late anaphase

La filiacion y la fecundacion "in vitro"

Las tecnicas de reproduccion asistida no solo representan una solucion para ayudar a superar problemas de esterilidad, sino que su practica conlleva problemas eticos y juridicos. Esta Tesis analiza los problemas que plantea la fecundacion "in vitro", desde el punto de vista de la filiacion, para determinar la paternidad y maternidad cuando se utilizan los gametos de la pareja o de un tercero. Desde este punto de vista, se estudian la situacion juridica del tercero -llamado donante- y de las madres subrogadas, asi como las acciones de filiacion Tambien se examina la problematica que plantea la congelacion de semen y embriones, al poder un hombre engendrar un hijo despues de muerto. Entre las fuentes que se analizan estan los principales informes extranjeros que han estudiado la problematica de estas tecnicas, asi como el Informe especial de..

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference

A miniaturized continuous dielectrophoretic cell sorter and its applications

There is great interest in highly sensitive separation methods capable of quickly isolating a particular cell type within a single manipulation step prior to their analysis. We present a cell sorting device based on the opposition of dielectrophoretic forces that discriminates between cell types according to their dielectric properties, such as the membrane permittivity and the cytoplasm conductivity. The forces are generated by an array of electrodes located in both sidewalls of a main flow channel. Cells with different dielectric responses perceive different force magnitudes and are, therefore, continuously focused to different equilibrium positions in the flow channel, thus avoiding the need of a specific cell labeling as discriminating factor. We relate the cells’ dielectric response to their output position in the downstream channel. Using this microfluidic platform that integrates a method of continuous-flow cell separation based on multiple frequency dielectrophoresis, we succeeded in sorting viable from nonviable yeast with nearly 100% purity. The method also allowed to increase the infection rate of a cell culture up to 50% of parasitemia percentage, which facilitates the study of the parasite cycle. Finally, we prove the versatility of our device by synchronizing a yeast cell culture at a particular phase of the cell cycle avoiding the use of metabolic agents interfering with the cells’ physiology