Bioactivity/cytotoxicity of micro-/nano-materials and novel development of fiber-optic probes for single cell monitoring

Manufactured nano-/micro-materials (MNMs) have been widely used and their interactions with niche biological environment are highly concerned for both of their biohazardous and bioactive effects, whereas no available comprehensive evaluations or regulations have been provided yet. This dissertation thus focuses on three major aspects: 1) fundamental toxicity understandings of a typical MNMs (zinc oxide nanoparticles), 2) bioactivity evaluations of representative bioactive MNMs, and 3) development of novel micro-probes for high spatial resolution monitoring. Firstly, the NP\u27s concentration, irradiation, hydrodynamic size, and the localized pH, ionic strength, NP zeta-potential as well as dissolved oxygen levels were found correlated with the production of hydroxyl radicals (•OH). Thus a novel physicochemical mechanism was hypothesized on •OH generation from ZnO NPs to cast light on cytotoxic mechanisms of MNMs. Secondly, silicate-/borate-based nano-/micro-sized glass fibers showed good rehabilitation capability and the underlying mechanisms were revealed as that quicker ion releasing and glass conversion (into hydroxyapatite (HA)) are the key to promote cell proliferation and migration, thus the wound-healing effect. Thirdly, several types of fiber-optic-based probes were developed to better cope with high spatial resolution, niche biological environmental detection. Up to date the best probe is able to acquire a pH resolution of up to ~0.02 pH unit within biologically relevant pH range of 6.17 - 8.11 with fast sensing time of ~5 seconds. Real-time monitoring of single live human cells were also demonstrated and validated in cytotoxic studies to detect early-onset of cell deterioration on different stages, indicating its powerful potent on studies that focusing on MNMs and single cells --Abstract, page iv

Contribution to the development of methods and systems for the automatization during the early stages of bioprocess development

This thesis is framed within the field of red biotechnology and more specifically in the development of bioprocesses for cell species that feature some therapeutical interest, either for the production of vaccines and monoclonal antibodies or stem cell experimental research. The main objective was the development and application of different instrumental techniques for the control and online monitorization of cell cultures. Oxygen consumption OUR (Oxygen Uptake Rate) was chosen as the central theme since this parameter has often been referenced as the most straighforward indicator of metabolic activity in animal cell culture. This thesis was carried out in the context of a Spin-Off project (Hexascreen Culture Technologies) whose objective was the development of disposable Minibioreactors intended for biopharmaceutical research. Obviously, this has led to a number of important trade-offs, as well as the proposal of several imaginative solutions to solve various technological challenges. For this reason and in order to offer a better idea of the work's scope, it was decided to include in the thesis not only the description of the method and results related to the OUR estimation but a detailed description of the systems developed. Results demonstrate the feasibility of a simplified procedure for estimating the oxygen consumption. This is a review of the Stationary liquid phase mass balance method which allows reducing the implementation cost and unlike the Dynamic method (The most usual thechnique) prevents changes on the oxygen tension that could affect the cell's normal arctivity. The proposed method is based on the accurate control of the oxygen concentration by means of PWM driven electrovalves and using the control loop internal signals to estimate the OUR.Aquesta Tesi doctoral està enquadrada en l'àmbit de la Biotecnologia vermella i més concretament en el desenvolupament de Bioprocessos relacionats amb espècies cel·lulars d’interès terapèutic, bé sigui per a la producció de vacunes, anticossos monoclonals o bé per a la recerca experimental amb cèl·lules mare. L'objectiu general ha estat el desenvolupament i aplicació de diferents tècniques instrumentals per al control i monitorització en línia de cultius cel·lulars, tant mateix d'entre les diferents tècniques emprades es va escollir la monitorització de la demanda d'oxigen O.U.R. (Oxygen Uptake Rate) com a tema central de la tesi degut a que aquest paràmetre ha estat referenciat sovint com un dels millors indicadors de l'activitat metabòlica en cultius de cèl·lules animals. Cal mencionar que la Tesi ha estat duta a terme en el context d'un projecte empresarial (HexaScreen Culture Technologies) l'objectiu del qual ha estat el desenvolupament de Minibioreactors d'un sol ús orientats al mon de la recerca Biofarmacèutica. Òbviament això ha comportant un número important de compromisos a l'hora d'abordar les diferents tasques, així com el plantejament de solucions imaginatives per a la resolució dels diferents reptes tecnològic. Per aquest motiu i per tal de transmetre una millor idea de l'abast del treball realitzat, es va decidir incloure en la tesi no només la descripció del mètode i resultats relacionats amb l'estimació de la O.U.R. sinó amés una descripció prou detallada dels sistemes desenvolupats. Pel que fa al tema central de la tesi, es demostra la viabilitat d'un procediment simplificat per a l'estimació de la demanda d’oxigen. Es tracta d'una revisió del procediment d'estimació de la OUR en condicions de concentració estacionària en la fase líquida que permet reduir-ne el cost de implementació tot prescindint de l'ús de cabalímetres màssics, així com a diferència del mètode dinàmic (Tècnica més habitual) evitar cap mena de canvi en la tensió d’oxigen que pogués afectar l’activitat normal de les cèl·lules. El mètode proposat, es basa en el control de la concentració d’oxigen mitjançant actuació PWM de les vàlvules d'aereació i l’ús dels propis senyals del llaç de control per tal d'estimar la O.U.R.Postprint (published version

Physiological Response Of The Giant Acorn Barnacle, Balanus nubilus, To Air Exposure

The giant acorn barnacle, Balanus nubilus, is a resident of the subtidal and low intertidal rocky shoreline on the Pacific Coast of North America (Alaska to Baja California). B. nubilusis notable for having the largest muscle fibers in the animal kingdom; fiber diameters that can exceed 3mm in adults! At such extreme sizes these muscle cells may be at risk for insufficient oxygen delivery to mitochondria owing to low SA:V ratios and long intracellular diffusion distances. Oxygen limitation to these muscles may be further exacerbated during low tide air exposure (emersion) or environmental hypoxia events, which are increasing in frequency and duration along the world’s coastlines. We are interested in characterizing the internal oxygen conditions of B. nubilus during air emersion and anoxia so that we can ultimately investigate the physiologic mechanisms by which B. nubilus maintains function in their giant muscle fibers during environmental oxygen limitation. To this end, we examined the effects of air emersion and anoxia on 1) hemolymph gas, pH and ion levels, 2) oxygen consumption rates (MO2; emersion only), and 3) respiratory behaviors (e.g., cirri beating). In the first experiment, we measured hemolymph pO2, pCO2, pH and ion ([Na+], [Cl-], [K+], [Ca2+]) concentrations at 0, 3, 6 and 9h of exposure to air emersion, anoxic immersion and normoxic immersion (control). Next, we compared the average MO2 of barnacles held in water and air for 6h at three common temperatures (10, 15, or 20°C) using intermittent (aquatic) and closed-system (air) respirometry. Lastly, we investigated the respiratory behaviors (% time operculum open, %time testing, % time pumping, % time cirri beating, cirri beat frequency, opercular pulse frequency) of B. nubilusduring acute (6h) exposure to air emersion, anoxic immersion and normoxic immersion (control). Our data revealed that hemolymph pO­2 was significantly decreased in the anoxic barnacles by 3h and remained significantly depressed relative to the normoxic control for 9h. The air-exposed barnacles, however, maintained hemolymph oxygen levels that were intermediate to the control and anoxia barnacles for the entire experiment, achieving levels that were significantly lower than normoxic barnacles only by 9h. We also found that oxygen consumption rates for B. nubilus held at ecologically realistic temperatures were similar in air and water. From these data we conclude that B. nubilus is relatively adept at taking up oxygen from the environment while out of the water, which is common for certain barnacle species, and that air emersion represents a relatively mild environmental stress for this species (at least from a gas-exchange perspective). Efficient aerial gas-exchange by the giant acorn barnacle is likely facilitated by seawater pools stored in the mantle cavity, which can directly take up oxygen from the air and make it accessible to soft tissues and gill-like structures on the inside of the shell. This strategy, however, would require complementary behaviors aimed at oxygenating the mantle cavity fluid (e.g, aperture opening, cirri extensions to facilitate mixing), and this is exactly what we see. In our behavior experiment we found that air-exposed barnacles (and, more surprisingly, anoxic barnacles) spent significantly more time with their cirri extended than our control animals, who engaged more in an aperture pumping behavior with their cirri retracted. These behavioral preferences existed even though there were no significant differences in the total time spent with their aperture open (regardless of the behavior occurring while open) between any of the treatments. There were also interesting findings in the ion data. While there were no significant treatment effects on [Na+], [Cl-], or [Ca2+], we did observe significantly higher [K+] by 6h in both the emersion and anoxic groups relative to the normoxic group. We predict that this change in [K+] is likely attributable to its role in acid-base buffering. There was a strong correlation between pCO2 levels and pH across all treatments; however, decreases in pO2 levels in the hemolymph, which corresponded with increases in pCO2 levels, had only minimal effects on the hemolymph pH, indicating a well-buffered system. In conclusion, we found that air exposure does not inhibit aerobic metabolism in B. nubilus, owing largely to efficient aerial oxygen uptake and perhaps also effective acid base-buffering. We therefore predict that muscle function would be preserved during periods of low-tide emersion. Anoxia, on the other hand led to a significant decline in hemolymph oxygen content, which suggests that environmental hypoxia is likely to diminish functionality of their giant muscle fibers. In a parallel study, we intend to investigate the plastic response of B. nubilus muscle fibers to the same conditions (air emersion and anoxic immersion)

The Challenges of O2 Detection in Biological Fluids: Classical Methods and Translation to Clinical Applications

Dissolved oxygen (DO) is deeply involved in preserving the life of cellular tissues and human beings due to its key role in cellular metabolism: its alterations may reflect important pathophysiological conditions. DO levels are measured to identify pathological conditions, explain pathophysiological mechanisms, and monitor the efficacy of therapeutic approaches. This is particularly relevant when the measurements are performed in vivo but also in contexts where a variety of biological and synthetic media are used, such as ex vivo organ perfusion. A reliable measurement of medium oxygenation ensures a high-quality process. It is crucial to provide a high-accuracy, real-time method for DO quantification, which could be robust towards different medium compositions and temperatures. In fact, biological fluids and synthetic clinical fluids represent a challenging environment where DO interacts with various compounds and can change continuously and dynamically, and further precaution is needed to obtain reliable results. This study aims to present and discuss the main oxygen detection and quantification methods, focusing on the technical needs for their translation to clinical practice. Firstly, we resumed all the main methodologies and advancements concerning dissolved oxygen determination. After identifying the main groups of all the available techniques for DO sensing based on their mechanisms and applicability, we focused on transferring the most promising approaches to a clinical in vivo/ex vivo settin

Novel sensors for neural glucose concentration based on enzyme fluorescent thin films fabricated on nanoparticle carriers using electrostatic layer -by -layer assembly

Currently, there is no means by which rapidly fluctuating glucose and lactate levels can be monitored simultaneously. This dissertation demonstrates that by combining the broad-band versatility of fluorescence spectroscopy with nanoassembly methods, it is possible to construct micro- and nanoscale sensors with precise composition and short diffusion length constant. The work is significant because of its potential as a platform for discovery of basic normal physiological processes that have previously been hidden from researchers\u27 views, more detailed studies of responses to drugs or other stimuli, and even clinical monitoring. The main goal of this work was to develop novel methods that enable the simultaneous study of glucose and lactate transients in the brain extracellular fluid. It is shown that on-line monitoring of glucose concentration can be accomplished using optical probes with nanoassembled analyte specific enzymes combined with fluorescent indicators. A model for fabrication was developed to predict the fluorescence spectrum for a given film architecture. A model for the coupled reaction-diffusion was developed to predict the oxygen concentration in the sensing region of the films as a function of glucose concentration. A model was developed to predict the resulting fluorescence spectrum for the infusion of nanoparticles of a given film architecture. Oxygen sensors were fabricated on quartz slides, optical fibers and nanoparticles. A protocol in vitro and in vivo sensor delivery was developed and the delivery are nanoparticle sensors to the dentate gyrus of the hippocampus was confirmed by real-time fluorescence monitoring of the infusion and fluorescence confocal imaging of sectioned rat brain tissue. The accuracy of the fiber probes was shown to be 0.5% for 0 to 100% oxygen. The fiber probes were further developed in to a glucose probe with the addition of GOx films and a coating of 100 μm PDMS coating, which served as a transport barrier to oxygen. The accuracy of the glucose probe was approximately 21% for 0 to 60 mg/dL glucose

Smart Medical Systems with Application to Nutrition and Fitness in Space

Smart medical systems are being developed to allow medical treatments to address alterations in chemical and physiological status in real time. In a smart medical system sensor arrays assess subject status, which are interpreted by computer processors which analyze multiple inputs and recommend treatment interventions. The response of the subject to the treatment is again assessed by the sensor arrays, closing the loop. An early form of "smart medicine" has been practiced in space to assess nutrition. Nutrient levels are assessed with food frequency questionnaires, which are interpreted by flight surgeons to recommend in-flight alterations in diet. In the future, sensor arrays will directly probe body chemistry. Near infrared spectroscopy can be used to noninvasively measure several blood and tissue parameters which are important in the assessment of nutrition and fitness. In particular, this technology can be used to measure blood hematocrit and interstitial fluid pH. The noninvasive measurement of interstitial pH is discussed as a surrogate for blood lactate measurement for the development and real-time assessment of exercise protocols in space. Earth-based application of these sensors are also described

EFFECTS OF FLOW RATE ON MESENCHYMAL STEM CELL OXYGEN CONSUMPTION RATES IN 3D BONE TISSUE ENGINEERED CONSTRUCTS CULTURED IN PERFUSION BIOREACTOR SYSTEMS

Bone grafts comprise a multibillion dollar industry, with over a million grafts occurring each year around the world. These grafts are commonly autologous or allogenic. Autologous grafts are taken from the patient, and are sourced from a donor site from the patient’s body. Allogenic grafts are derived from a cadaver donor. Each of these graft types are associated with issues such as donor site morbidity in autologous grafts and immunological response in allogenic grafts. Bone tissue engineered constructs are a logical approach to combat the issues commonly encountered with current autologous and allogenic bone grafting techniques. While it is possible to grow bone tissue engineered constructs in vitro, it is necessary to destroy the construct to determine the number and type of cells present. Recent work has led to the development of models that have the potential to predict the number and types of cells within the construct through metabolite monitoring. The metabolites, such as glucose and oxygen, are used to monitor cell growth while the construct remains in the bioreactor, eliminating the need to lyse cells and destroy the scaffold. This allows for the development of quality assurance methodologies that are explicitly utilized on the specific graft to be used on a patient. These models, however, neglect to account for factors affecting the construct, such as flow rate and scaffold geometry. This study aimed to determine the flow characteristics present within the bioreactor system, and hoped to quantify the effects of these characteristics on oxygen uptake rates of mesenchymal stem cells. The work done utilized a residence time distribution analysis using an easily monitored dye to develop residence time distribution functions, and associated these functions with literature values to characterize the flow patterns and residence times of media moving through the construct. Findings indicate that flow within the bioreactor system is well approximated by linear tubular flow reactors, associated with gradients in the radial direction at low flow rates. Additionally, oxygen uptake rates of stem cells in these conditions have a strong linear correlation with residence times of media in the cassette, providing the data needed to develop a predictive model for oxygen uptake rates. These models also show promise for providing corrective functions for on-line oxygen monitoring systems in current bioreactor designs

Nanoparticle-based intra-cellular diagnostics

An in-depth understanding of biochemical processes occurring within the cell is a key factor for early diagnosis of disease and identification of appropriate treatment. Intracellular sensing using fluorescent nanoparticles (NPs) is a potentially useful tool for real-time, in vivo monitoring of important cellular analytes. This work is focused on synthesis of organically modified-silicate (ORMOSIL) optical nanosensors for the quantitative analysis of oxygen concentration and pH sensing inside the cell. The structure of the sensor consists of a biofriendly silica matrix with encapsulated oxygen/pH-sensitive dyes. The optical probes used in this work are the oxygen-sensitive ([Ru(dpp)3]2+) complex and pH-sensitive fluorescein isothiocyanate (FITC) coencapsulated with the ATTO488 and Texas Red as the reference dyes, respectively. In order to obtain silica-based NPs, the Stöber method was used. The NPs were characterised using techniques such as Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), fluorescence and other spectroscopic techniques. The second part of this work focuses on the introduction of the NPs into the cell and intracellular sensing. In this work the oxygen and pH nanosensors are introduced in a number of established human and mouse cell lines. Internalization of NPs within the cell is investigated using fluorescence confocal microscopy techniques. The detection of the optical signal is based on both ratiometric and fluorescence lifetime – based measurements carried out on the wide-field and confocal microscopes with fluorescence lifetime imaging platforms. After the NP calibration, the response of the cell to the different extracellular oxygen concentration is investigated. Oxygen and pH sensing is the starting point for this intracellular diagnostics research. The silica-based NPs, thanks to the flexible processing conditions, allow for tailoring of pore size and hydrophilic-hydrophobic balance. The possibility to control these two parameters makes the NPs a very promising tool for a better understanding of many processes in living cells