Improving the function of islet and beta-cell grafts

Background: Human islet transplantation would offer a less invasive and more physiological alternative than whole pancreas transplantation and insulin injections respectively for the treatment of diabetes mellitus if islet graft survival can be improved. Initial recipient post-transplant insulin independence declines to <10% after 5 years. Factors contributing to graft failure include enzymatic disruption of the islet microenvironment during isolation, diabetogenic effects of immunosuppressants and metabolic stress resulting from slow revascularisation. Aims: To investigate the effect of co-culture in both static (SC) and rotational culture (RC) of BRINBDII beta-cells (Dl1) and human umbilical vein endothelial cells (HUVEC) on Dl1 insulin secretion; and the effect of a thiazolidinedione (TZD) on DII function and HUVEC proliferation. To assess the effect of culture media, SC, RC and a TZD on human islet morphology, insulin secretion and VEGF production. To initiate in vivo protocol development for assessment of revascularisation of human islet grafts. Methods: D11 cells were cultured +/-TZD and co-cultured with HUVEC +/-TZD in SC and RC. Dl1 insulin secretion was induced by static incubation with low glucose (1.67mM), high glucose (l6.7mM: and high glucose with 10mM theophylline (G+T) and determined by ELISA. HUVEC were cultured +/-TZD in SC and RC and proliferation was assessed by ATP luminescence assay and VEGF ELISA. D II and HUVEC morphology was determined by immunocytochemistry. Human islets were cultured in SC and RC in various media +/-TZD. Insulin secretion was determined as above and VEGF production by fluorescence immunocytochemistry (FI) and ELISA. Revascularisation of islet grafts was assessed by vascular corrosion cast and FI. Results: Dll cultures showed significantly increased insulin secretion in response to 16.7mM and G+T over basal; this was enhanced by RC and further improved by adding 10mM TZD. Untreated Dll/HUVEC co-cultures displayed significantly increased insulin secretion in response to 16.7mM and G+T over basal, again enhanced by RC and improved with 10mM TZD. 10mM TZD significantly increased HUVEC proliferation over control. Human islets maintained in medium 199 (mI99) in SC and RC exhibited comparable maintenance of morphology and insulin secretory profiles compared to islets maintained in RPMI, endothelial growth media and dedicated islet medium Miami# I. All cultures showed significantly increased insulin secretion in response to 16.7mM and G+T over basal; this was enhanced by RC and in certain instances further improved by adding 25mM TZD. TZD increased VEGF production and release as determined by ELISA. Post-implant vascular corrosion casts of mouse kidneys analysed by x-ray micro tomography indicates a possible TZD enhancement of microvessel growth via VEGF upregulation. Conclusions: D II /HUVEC co-culture in SC or RC does not alter the morphology of either cell type and supports D 11 function. TZD improves 0 I I and D I I/HUVEC SC and RC co-culture insulin secretion while increasing HUVEC proliferation. Human islet RC supports islet functional viability and structural integrity compared to SC while the addition of TZD occasionally further improves secretagogue induced insulin secretion. Expensive, 'dedicated' islet media showed no advantage over ml99 in terms of maintaining islet morphology or function. TZD upregulates VEGF in islets as shown by ELISA and suggested by x-ray micro tomography analysis of vascular corrosion casts. Maintenance of islets in RC and treatment with TZD prior to transplant may improve the functional viability and revascularisation rate of islet grafts.

Femtosecond laser micro-inscription of optical coherence tomography resolution test artifacts

Optical coherence tomography (OCT) systems are becoming more commonly used in biomedical imaging and, to enable continued uptake, a reliable method of characterizing their performance and validating their operation is required. This paper outlines the use of femtosecond laser subsurface micro-inscription techniques to fabricate an OCT test artifact for validating the resolution performance of a commercial OCT system. The key advantage of this approach is that by utilizing the nonlinear absorption a three dimensional grid of highly localized point and line defects can be written in clear fused silica substrates.

Advances in femtosecond micromachining and inscription of micro and nano photonic devices

This thesis has focused on three key areas of interest for femtosecond micromachining and inscription. The first area is micromachining where the work has focused on the ability to process highly repeatable, high precision machining with often extremely complex geometrical structures with little or no damage. High aspect ratio features have been demonstrated in transparent materials, metals and ceramics. Etch depth control was demonstrated especially in the work on phase mask fabrication. Practical chemical sensing and microfluidic devices were also fabricated to demonstrate the capability of the techniques developed during this work. The second area is femtosecond inscription. Here, the work has utilised the non-linear absorption mechanisms associated with femtosecond pulse-material interactions to create highly localised refractive index changes in transparent materials to create complex 3D structures. The techniques employed were then utilised in the fabrication of Phase masks and Optical Coherence Tomography (OCT) phantom calibration artefacts both of which show the potential to fill voids in the development of the fields. This especially the case for the OCT phantoms where there exists no previous artefacts of known shape, allowing for the initial specification of parameters associated with the quality of OCT machines that are being taken up across the world in industry and research. Finally the third area of focus was the combination of all of the techniques developed through work in planar samples to create a range of artefacts in optical fibres. The development of techniques and methods for compensating for the geometrical complexities associated with working with the cylindrical samples with varying refractive indices allowed for fundamental inscription parameters to be examined, structures for use as power monitors and polarisers with the optical fibres and finally the combination of femtosecond inscription and ablation techniques to create a magnetic field sensor with an optical fibre coated in Terfenol-D with directional capability. Through the development of understanding, practical techniques and equipment the work presented here demonstrates several novel pieces of research in the field of femtosecond micromachining and inscription that has provided a broad range of related fields with practical devices that were previously unavailable or that would take great cost and time to facilitate.

Fabrication of high quality optical coherence tomography (OCT) calibration artefacts using femtosecond inscription

Optical coherence tomography (OCT) is a non-invasive three-dimensional imaging system that is capable of producing high resolution in-vivo images. OCT is approved for use in clinical trials in Japan, USA and Europe. For OCT to be used effectively in a clinical diagnosis, a method of standardisation is required to assess the performance across different systems. This standardisation can be implemented using highly accurate and reproducible artefacts for calibration at both installation and throughout the lifetime of a system. Femtosecond lasers can write highly reproducible and highly localised micro-structured calibration artefacts within a transparent media. We report on the fabrication of high quality OCT calibration artefacts in fused silica using a femtosecond laser. The calibration artefacts were written in fused silica due to its high purity and ability to withstand high energy femtosecond pulses. An Amplitude Systemes s-Pulse Yb:YAG femtosecond laser with an operating wavelength of 1026 nm was used to inscribe three dimensional patterns within the highly optically transmissive substrate. Four unique artefacts have been designed to measure a wide variety of parameters, including the points spread function (PSF), modulation transfer function (MTF), sensitivity, distortion and resolution - key parameters which define the performance of the OCT. The calibration artefacts have been characterised using an optical microscope and tested on a swept source OCT. The results demonstrate that the femtosecond laser inscribed artefacts have the potential of quantitatively and qualitatively validating the performance of any OCT system.