| Summary: | Stem cells serve as important models for the in vitro study of many in vivo micro-environments. Their function relies largely on the highly ordered organisation of cells, matrices and soluble factors within defined architectures. Therefore, the ability to manipulate microscopic objects through precise in vitro approaches provides a new means to replicate these complex arrangements and give insights into factors that influence biological development. In this study, the progression of specific patterning technologies that allow precise control over cell position at the micron scale has been investigated. Initial studies demonstrated a stencilling based micro-patterning technique, which utilised aerosol deposition of a chemical agent, to induce region selectivity and define cell adhesive areas on a poor cell responsive surface. Micro-stencils acted as a mask to spatially restrict l,l'-Carbonyldi imidazole (CDI) aerosols to poly(2-hydroxyethyl methacrylate) (polyHEMA) coated surfaces, to which fibronectin was subsequently immobilised. Surface characterisation analysis revealed that a change in surface topography and a decrease in surface hydrophilicity did not negatively affect the adhesion and proliferation of cells. NIH-3T3 cells were seeded onto micro-patterns in defined medium conditions where cell proliferation, using the MTS assay, was assessed over time. The combined use of polyHEMA substrates with this functionalisation technique produced persistent patterns that were preserved for up to 96 hours. This method achieved well defined patterns of cells over macro scales with prospective uses in future tissue engineering applications. Secondly, an optical based micro-patterning technology, utilising a bespoke holographic optical tweezer (HOT) system, was used to precision build cellular micro-architectures. N1H-3T3 cells and mouse embryonic stem (mES) cells were patterned using an infrared laser. Multiple optical traps were generated by laser reflection off a spatial light modulator (SLM). The reflected light was focused through a high resolution numerical aperture objective lens to produce a defined three dimensional (3D) pattern (the hologram). PolyHEMA was used to prevent substrate stickiness prior to pattering. Opening work of this technique patterned two dimensional (2D) cell arrangements that were retained using the developed micro-stencilling method to monitor subsequent responses of cell viability, proliferation and differentiation. LIVE/DEAD® staining and proliferation assessment using the Click-iT® EdU assay demonstrated that the laser caused no obvious damaging effects on the cells. Immunocytochemical staining of Brachyury, Nestin and GATA-4 showed differentiation of mesoderm and ectoderm but no endoderm expression of small scale structures (> 4 cells). Similarly positive Oct-3/4 expression showed evidence of undifferentiated cells. The next part of the study went on to demonstrate the stabilisation of patterned cell structures within a temperature or enzyme controlled hydrogel support network to retain 3D architectures. Complex co-cultures of cells and poly(lactic-co-glycolic acid) (PLGA) polymer materials were also investigated to reproduce analogues of the cells physical environment. This manipulation tool attained single cell resolution capable of manipulating microscopic arrangements of cells with future applications within the fields of developmental and stem cell biology. It is hoped that the engineering of 3D biological structures in vitro will allow for the generation of more complex and representative cell models with greater definable and tuneable characteristics to assess stem cell fate in the future.
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