Overall hypothesis:

Cell-mediated metabolic gradients within three dimensional cellular structures control the development of spatial patterns of cell viability and differentiation

Objectives:

• To develop and optimise micro-environmental monitoring technologies to assess temporal and spatial gradients of oxygen, glucose and lactate
• To examine the behavioural responses of selected cells to various in vitro 3-D environments

Background:

Oxygen and nutrient requirements and the limits of diffusion induce the formation of spatial gradients of key nutrients and metabolites such as oxygen, glucose and lactate in tissues. Low oxygen tensions enhance stem cell survival and stem cell self-renewal and immortality may depend on anaerobic respiratory pathways. The proposed study will analyze micro-environmental factors related to stem cell survival and the patterns of intra-cellular metabolic activity associated with stem cell self-renewal. The study is expected to provide data of interest both to biological problems and to clinical problems of stem cell targeting. The micro-environmental milieu will be characterised, using novel, non-destructive analytic techniques, to predict spatial and temporal profiles of cell survival and differentiation within a 3-D environment. Development of monitoring technologies: Multiphoton Fluorescence Life-Time Imaging (FLIM) techniques will be adapted to probe the spatial and temporal characteristics of the oxygen microenvironment within 3D constructs. FLIM is based upon fluorescence which occurs when molecules excited by light absorption revert to their original state by light emission. Critically, fluorescence occurs over a very brief time period, typically in the picosecond to nanosecond range, and the precise kinetics of this radiative and non-radiative decay depends on the local environment. By combining this technique with simultaneous live cell confocal microscopy it will be possible to correlate the oxygen microenvironment with changes in cell activity and associated intracellular signalling pathways. FLIM analysis will be performed using a Leica TCS SP2 confocal scanning microscope adapted to provide multiphoton capability enables deep penetration into the sample with minimal photobleaching or phototoxicity. In vitro assessment of stem cell survival and differentiation within 3-D systems: Appropriate in vitro experimental models will be developed and tested. A number of parameters will be varied to influence pO2 and glucose microenvironmental conditions, thereby influencing spatially-defined patterns of cell survival and differentiation. Depending on the results obtained from assays of experimental transplant sites (see below) other pO2 values may also be evaluated. The experimental design, involves a large number of variable/permutations. The experimental design will be subjected to a Taguchi-type methodology to reduce the experimental workload to manageable levels. Constructs will be maintained in culture for up to 21 days prior to analysis of spatial profiles of cell survival, cell metabolic response, oxidative DNA damage and phenotype.