Development and in-vitro evaluation of a potentially implantable fibre-optic glucose sensor probe

Type I diabetics need regular injections of insulin to survive. Insulin allows the cells of the body to extract glucose from the blood supply to use as fuel. Without insulin the cells turn to other backup fuel sources,this can cause side effects that are quickly fatal or gradual wasting of the bodies tissues. The use of insulin, however, is not danger free, as an incorrect dosage can quickly lead to the reduction of glucose circulating in the blood to drop to a dangerously low level. Without glucose circulating in the blood supply the brain quickly runs out of fuel causing coma and death. Because of this, a means to constantly monitor blood glucose levels has been sought for the last two decades. With such a device, diabetics could judge the correct amount of insulin to inject and be warned of low blood glucose levels. However, to date no reliable portable system has been produced. Recent developments in fibre optic biosensor technology, suggested a possible route to achieves this goal. The work in this thesis presents the development and testing of such a sensor. The sensor presented in this thesis is based around a commercial fibre optic blood gas sensor, the Paratrend 7. The oxygen-sensing element of this device was modified into a glucose sensor using polymer membranes incorporating the enzymes glucose oxidase and catalase. The research was aimed at building a glucose sensor that could be developed into a working blood glucose sensor in the minimum amount of time if the research proved successful. For this reason the Paratrend 7 sensor system was chosen to provide a clinically tested sensor core around which the glucose sensor could be built. The initial experiment, which used a Paratrend7 sensor coated in polyHEMA and glucose oxidase, produced a sensor of diameter of 700µm with a range of 0 to 4mM/1 of glucose and a 90% response time of <100 seconds in a solution with a 15% oxygen tension. The sensor design was then developed to incorporate the enzyme catalase to protect the glucose oxidase and an outer diffusion limiting polyHEMA membrane. This produced a sensor with a range of 0 to 6 mM/l and a response time of <100 seconds. The method of coating the sensors was'then improved, through a series of stages, until an optomised dip coating technique was developed. This technique produced sensors with ranges (in 7.5KPa oxygen tension solutions) between 0 to 3mM/l and 0 to lOmM/1, responsetimes of <100 seconds in some cases and with diameters of 300µm. By using a partial polyurethane outer coat the range of the sensors was increased form 0 to 4mM/l up to 0 to 24mM/1, in one case, with 90% response times in the 100to 500 second range. The sensors were then sterilised using gamma radiation and their performance before and after sterilisation examined. The gamma sterilisation was found to cause a reduction in the range of the sensors,for example 0 to 24 m /I down to 0 to 14mM/l in one case. The affect of 24 hour operation in a 5mM/1 solution of glucose and storage, for up to three months, was then investigated. Both processes were found to reduce the operational range of the sensors,0 to 20 reduced to 0 to 15 mM/i, in one case,for 24 hour operation and form 0 to 15mM/1 reduced to 0 to 11mM/1in one case for a storage time of three months. The use of the enzymes glucose oxidase and catalase together in a fibre optic as can sensor has not been previously reported in the literature as far be ascertained. The comparison of sensor performance before and after gamma sterilisation also appears to be unique as does the gamma sterilisation of a fibre optic glucose sensor

Development of a non-invasive method to detect pericellular spatial oxygen gradients using FLIM

PhDExtracellular oxygen concentrations affect cellular metabolism and influence tissue function. Detection methods for these extracellular oxygen concentrations currently have poor spatial resolution and are frequently invasive. Fluorescence Lifetime Imaging Microscopy (FLIM) offers a non-invasive method for quantifying local oxygen concentrations. However, existing FLIM methods also show limited spatial resolution >1 μm and low time-resolved accuracy and precision, due to widefield time-gate. This study describes a new optimised approach using FLIM to quantity extracellular oxygen concentration with high accuracy (±7 μmol/kg) and spatial resolution ( ≅ 0.3 μm). An oxygen sensitive fluorescent dye, tris(2,2′-bipyridyl)ruthenium(II) chloride hexahydrate [Ru(bipy)3]+2, was excited with a multi-photon laser and fluorescence lifetime was measured using time-correlated single photon counting (TCSPC). The system was fully calibrated with optimised techniques developed for avoiding artefacts associated with photon pile-up and phototoxicity, whilst maximising spatial and temporal resolution. An extended imaging protocol (1800 sec) showed no phototoxic effects on cells at dye concentrations of <0.4 mM. Extracellular spatial oxygen gradients were identified around isolated chondrocytes, seeded in three-dimensional agarose gel. The technique was validated by regulating oxygen cellular consumption and thus confirming that the oxygen gradient was governed by cellular consumption. The technique identified a subpopulation of cells exhibiting statistically significant spatial oxygen gradients at the cell perihery. The subpopulation was shown to be significantly larger in cell diameter correlating with what that expected from chondrocytes in the deep zone. This technique provides an exciting opportunity to non-invasively quantify pericellular spatial oxygen gradients from within three-dimensional cellular constructs without prior manipulation of the cells. Thus by examining cellular metabolisms it will advance our understanding of the optimal cellular environment for tissue engineering and regenerative medicine

Mathematical Modelling of Oxygen and Glucose Conditions for Mesenchymal Stem Cells in Culture and Bone Marrow

EngD ThesisThe prevalence of 3D tissue culture systems is increasing in order to overcome the perceived limitations of 2D culture in terms of providing a biomimetic niche for cells. However, the move to 3D systems requires that the availability of nutrients and oxygen within a 3D system is understood. The aim of this project was to develop mathematical models which would allow conditions in 2D and 3D culture and in vivo to be better understood. A mass transfer model was developed using physical data from experiments, the conservation of mass and Fick’s law of diffusion, using the 2D and 3D culture of mesenchymal stromal cells as an exemplar system. The model was then used to create oxygen and glucose profiles in 2D and 3D (spheroids and suspension) culture to provide a basis for comparison between the different systems. Predicted mass transfer of oxygen was found not to be affected by spheroid culture when compared to 2D culture, however mass transfer of glucose was restricted creating significant glucose concentration gradients through the spheroids. The predicted profiles in spheroid culture were applied to other culture systems with the aim of inducing the changes observed in the mesenchymal stromal cells in spheroid culture Altered glucose concentrations were not sufficient to induce dedifferentiation in 2D adherent mesenchymal stromal cells nor result in the same cell size decrease as seen in 3D spheroid culture. Using suspension cultures, a comparable size decrease to 3D spheroid culture was observed. Mass transfer modelling of the in vivo mesenchymal stromal cells environment in bone marrow was also developed to compare the in vitro culture conditions to the natural environment. It is concluded that oxygen concentrations within cell culture are lower than in bone marrow but relatively stable in the culture systems modelled. Glucose concentrations are significantly reduced within spheroid culture

The role of HIF-1 alpha in the localization of embryonic stem cells with respect to hypoxia within teratomas

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2005.Includes bibliographical references (leaves 172-183).In embryonic stem (ES) cell tumors, the hypoxia-inducible transcription factor, HIF- 1[alpha], has been shown to be a tumor suppressor, and HIF-1[alpha]-expressing cells have been shown to localize preferentially in vivo to regions near tumor vasculature. These differences were proposed to be due to increased hypoxia-induced apoptosis and growth arrest of HIF-1[alpha]-expressing ES cells. This thesis presents a careful investigation into the localization of ES cells in vitro and in vivo with respect to hypoxia. A sandwich culture system was utilized in which controlled gradients of oxygen and nutrients are developed in the vicinity of the tumor cells. A diffusion-consumption model was utilized to predict the oxygen and glucose concentration profiles within the system. Oxygen and glucose consumption rates were measured and used as inputs into the model, and the concentration profiles were found to depend on a single experimental parameter, the cell density within the system. The optimum cell density was found in which stable, measurable oxygen gradients develop over 2-3 mm. The model demonstrated excellent agreement between the predicted oxygen concentration profiles and experimentally determined oxygen gradients. In vitro, there was no difference in localization with respect to hypoxia between tumor cells expressing or lacking HIF-1[alpha].(cont.) In addition, there was no difference in apoptosis, proliferation, or migration of the tumor cells in vitro based on HIF-1[alpha] expression. Likewise, a quantitative study on localization of tumor cells within tumors in vivo demonstrated no difference between localization of HIF-1[alpha]-expressing vs. HIF-1[alpha]-lacking ES cells within tumors with respect to blood vessels or hypoxia. These results differ from previous studies, perhaps due to clonal variation of the cell phenotype or the interplay of other complex environmental factors that were not considered in this study. Interestingly, the HIF-1[alpha]-lacking cells were found to exhibit increased tumor growth relative to the HIF-1[alpha]-expressing cells, perhaps due to a normalization of the blood vessels within the HIF-1[alpha]-lacking tumors. These studies reveal the complex role of HIF-1[alpha] in tumor growth and tumor cell localization, as well as develop a useful quantitative experimental model for studying the role of the microenvironment in tumors or in embryonic stem cell biology.by David M. Cochran.Ph.D

Seeing Through the Fog: Using Scattered Light to Peer Deeper into Biological Tissue

Optical scattering is a fundamental problem in biomedical optics and limits most optical techniques to shallow operating depths less than 1 millimeter. However, although the scattering behavior of tissue scrambles the information it contains, it does not destroy it. Therefore, if you can unscramble the scattered light, it increases the accessible imaging depths up the absorption limit of light (several centimeters deep). One such way to beat optical scattering is using wavefront shaping. Borrowing ideas from adaptive optics in astronomy and phased arrays in radar and ultrasonic imaging, the basic concept of wavefront shaping is to control the phase and amplitude of the light field in order to harness scattered light. Using wavefront shaping techniques, scattered light can be used to form focal spots or transmit information through or inside optically scattering media. Furthermore, even without correcting for scattering directly by shaping the input light field, the properties of the scattered light can be analyzed to recover information about the structure and dynamic properties of a sample using methods from diffuse optics. The main contributions of this thesis are along these two lines of research: moving wavefront shaping toward more practical applications and developing new techniques to recover useful physiological information from scattered light. This is developed through three main projects: (1) an investigation of how dynamic samples impact the scattering process and the practical implications of these dynamics on wavefront shaping systems, (2) the development of a wavefront shaping system combining light and ultrasound to focus light inside acute brain slices to improve light delivery for optogenetics, (3) a novel method to sensitively detect the dynamics of scattered light and use it to tease out information about the flow of blood within the tissue sample of interest.</p

37th Rocky Mountain Conference on Analytical Chemistry

Final program, abstracts, and information about the 37th annual meeting of the Rocky Mountain Conference on Analytical Chemistry, co-sponsored by the Colorado Section of the American Chemical Society and the Rocky Mountain Section of the Society for Applied Spectroscopy. Held in Denver, Colorado, July 23-27, 1995

40th Rocky Mountain Conference on Analytical Chemistry

Final program, abstracts, and information about the 40th annual meeting of the Rocky Mountain Conference on Analytical Chemistry, co-sponsored by the Colorado Section of the American Chemical Society and the Rocky Mountain Section of the Society for Applied Spectroscopy. Held in Denver, Colorado, July 25 - August 1, 1998