Development of advanced 3D tissue models and O2 imaging methodologies
Loading...
Files
Full Text E-thesis
Date
2016
Authors
Jenkins, James
Journal Title
Journal ISSN
Volume Title
Publisher
University College Cork
Published Version
Abstract
This thesis presents the development and evaluation of new luminescent sensors and probes, optimisation of the tumour spheroid model and the various applications for these in vitro tissues combined with novel O2 and temperature nanoparticle probes. Here a new method for O2 sensing was introduced, measuring extracellular oxygenation using solid state polystyrene scaffold (AlvetexTM) impregnated with the phosphorescent dye PtTFPP. We accessed the toxicity, sensitivity and photostability of the O2-sensitive scaffolds and monitored cell oxygenation following seeding with both PC12 and HCT116 cells. Using TCSPC and PLIM multiplexed with fluorescent markers and fluorescent staining we could non-invasively correlate lifetime values with toxicity and drug stimulation. Several novel O2 and temperature-sensitive nanoparticle probes were evaluated. These probes displayed sufficient brightness, photostability and sensitivity. Furthermore, they showed minimal toxicity and could penetrate 3D tumour spheroids in depth, showing efficient staining and even distribution. We applied the imaging methodology to the 3D spheroid model, investigating which method of formation from "free floating", "hanging drop" and LipidureTM, produced the most uniform, viable and metabolically active in vitro tissue. Numerous applications were improved and aided by the new O2 measuring platforms. Combining the novel probes with our new 3D models we could monitor the effects of chemotherapeutic drugs in both seeded O2 scaffolds and 3D spheroids. We demonstrate the application of FLIM method for multi-parametric analysis of O2 simultaneously with temperature and confirm the existence of temperature gradients in the 3D cell-based model. Finally, we applied the O2 probe and its measurement via 2 PLIM method to elucidate the function of SPCA2 in human colon cancer HCT116 cells, grown in ambient and decreased O2 levels. We could correlate SPCA2 upregulation with hypoxia in both monolayer and in spheroids. Furthermore, we discovered that SPCA2 is up-regulated by cell density, playing a role in Mn2+ transport and cell cycle progression in cancer cells. Results show that the developed probes and techniques provide a useful tool for the highly sensitive imaging of intracellular and extracellular O2, temperature and other important parameters.
Description
Keywords
Hypoxia , Nanoparticle probes , 3D tissue models
Citation
Jenkins, J. 2016. Development of advanced 3D tissue models and O2 imaging methodologies. PhD Thesis, University College Cork.