Tesi di Dottorato

The PhD project is based on the applications of several x-ray microscopy techniques for compositional and morphological studies at nanoscale spatial resolution to a biological problem, i.e. the quantitative determination of morphological and compositional properties of epithelial cells. Three x-ray microscopy techniques were exploited in this work: X-ray fluorescence microscopy, X-ray phase contrast imaging and nanotomography, which were made at the ID16NI beamline of the European Synchrotron Radiation Facility in Grenoble, France. In addition to synchrotron-based techniques, also Atomic Force Microscopy was performed. The latter was used for morphology characterization, and forcalibration and comparison purpose. The main aim of this study was to quantitatively determine the map of iron concentration at nanoscale spatial resolution of epithelial cells infected by bacterial pathogens in the presence or absence of lactoferrin (Lf), an iron-chelating glycoprotein of natural immunity. Two experiments have been carried out at ESRF, one on freeze dried cells, and one on frozen hydrated cells this last using the cryo stage foreseen in the Id16 NI beamline, in order to examine cells as close as possible to their native state, and to avoid radiation damage. The measurement and data analysis protocols have been carefully studied for optimal combination of all the techniques, to give quantitative results. Iron concentration and mass fraction maps have been obtained, which give an insight about the modification of iron spatial distribution under the influence of lactoferrin. Moreover, for the first time it has been demonstrated the possibility to obtain quantitative element concentration in cells through the combination of x-ray nanotomography in phase contrast and x-ray fluorescence microscopy.

On the global scale, liver diseases are severe public health problems, with the incidences of end-stage liver disease (ESLD) rising annually. Isolated hepatocytes represent a good model of liver metabolism because they are able to perform the full range of functions. In recent years, biochemical and biotechnological engineering have been applied to the culture of human and animal hepatocyte cells, which requires the design, operation, and control of complex appropriate bioreactors. In this work, the predictable, stable and durable operation of two types of bioartificial reactors for cell cultures is investigated. The thesis is divided into the following two parts. Part I: Fluidized bed bioreactor Fluidized-bed-based biomedical devices acting as bioartificial liver, in which cells are trapped and encapsulated into appropriated fluidized beads, have proved effective solutions to many respects. However, the bioreactor performance is significantly affected by the hydrodynamics and mass transfer, not well characterized yet for most aspects. In the present work, the intrinsic and fluidization properties of alginate beads as encapsulation medium for hepatic cells are carefully analyzed experimentally using two rigs at different scales. Appropriate alginate beads were prepared and characterized in terms of size distribution and density. Expansion properties were evaluated for free alginate beads (i.e. without hepatic cells) using saline (Ringer) solutions as fluidization medium. Bed expansion tests over a wide range of voidage values have been conducted in a 1-cm diameter column, used for perfusion during in vitro experiments, as well as in a 10-cm diameter column close to human size bioreactor, in the latter case at two temperatures: ambient (20°C) and human body (37°C) conditions. Full fluid-dynamic characterization of the alginate beads is conducted, including expansion data, terminal velocity measurements, and velocity-voidage plots and their elaboration in terms of Richardson-Zaki parameters. Part II: Hollow fiber membrane bioreactor Due to their structure affine to the physiological environment in vivo, hollow fibre membrane bioreactors in crossed configuration can provide favourable conditions for the cell behaviour and metabolism. Specific devices have been proposed in recent years with very promising potential for applications. To be able to develop bioartificial systems that operate effectively and for the long term, in addition to handling the biological complexities, fluid dynamics and transport phenomena require an advance model, careful control, and appropriate automation strategies. Tight control of the culturing environment and strategies for dealing with some inherently unsteady changes of conditions in a membrane bioreactor is investigated by developing and implementing a new hydrodynamic dual control system for an existing bioreactor prototype. The experimental implementation of the sensors-controllers-actuators system is complemented by the development of a transient mathematical model of the instrumented bioreactor, in which the membrane unit is treated as a three-compartment model. A four-input/seven-state transient model of the bioreactor is obtained, able to describe the time evolution of the flowrates, the extra-capillary space liquid level and the oxygen concentration across the system. The selection of appropriate sensors and the manipulated control variables is discussed. Bioreactor dynamic simulation and control is carried out within the Matlab/Simulink environment and Matlab is also used as a platform for the experimental data digital acquisition and control logic implementation (e.g. controller tuning), allowing both for flexibility with testing of different control schemes and for direct comparison of simulated and experimental values. Different experiments with selected input changes were carried out under idealized conditions and using water as perfusing medium. The applied stimuli served to mimick causes of previously observed bioreactor malfunctions (e.g. high sensitivity to liquid level variations during prolonged cell culturing experiments) and check the control system efficacy and efficiency. Finally, the developed control system is utilized during a prolonged experiment of multi-cell culture within the membrane bioreactor, demonstrating the reliable, continuous and successful cultivation for nearly one month time. The set of results collected during the present work allows to achieve new insight into the operation and reliability of bioreactors for application as bioartificial devices, by improving the capacity to predict their behaviour and better design their structure as well as by enhancing the control over the cell culture environment conditions

Tesi di Dottorato

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