A cell’s interaction with its extracellular environment is critical to tissue structure and function. This work investigates the effect of substrate topography on selective cell adhesion and morphology. Alterations in cell response to micro- and nanoscale signals and cues can cause changes in downstream functions of proteins and complexes such as invasive and metastatic motility of malignant tumour cells and the differentiation direction of stem cells. Biomaterial surfaces can be modified to provide different chemical and topographical cues and encourage controlled cell-substrate interaction. At the protein level, template substrates have shown and increased affinity for selective binding of the imprinted antigen or antibody. Topography of a cell’s microenvironment may be replicated as a permanent polymer mould by bioimprinting technology, which was developed at University of Canterbury. The resulting high resolution methacrylate polymer samples have been used for imaging and analysis, but have not previously been investigated as cell culture substrates. This work investigates the effect of bioimprint and photolithographic substrate patterning on cell behaviour in culture. Optimisation of a methacrylate co-polymer resulted in a 6:3:1 ethylene glycol dimethacrylate: methacrylic acid: photoinitiator polymer mixture cured by 240 seconds of UV exposure. The polymer was used to replicate cell membrane features into a permanent polymer mould [a bioimprint]. The resulting high resolution methacrylate bioimprints were cleaned and sterilised for use as a secondary cell culture substrate. Ishikawa endometrial cancer cells were cultured on bioimprinted methacrylate polymer substrates. Preliminary results showed preferential cell adhesion to bioimprinted areas over flat areas and also showed three dimensional spheroid growth instead of lateral two dimensional monolayer spreading. At higher seeding densities, preferential adhesion was similarly noted as well as peeling artefacts of shear stresses and cell size variation on flat methacrylate substrate regions. Fluorescent imaging and cell culture stencilling highlighted the association of secondary cells with bioimprint substrate features. To determine whether preferential cell adhesion effects were due to bioimprint features or general topography modification, secondary cancer cells were cultured on comparable photolithographically-defined, geometrically-patterned substrates. Methods for transferring regular pattern arrays into methacrylate polymer substrates were developed. No organisation or preferential adhesion effects were observed in association with pillar and hole patterns between 5-30 µm. However, artefact incidence in methacrylate polymer replication techniques led to development and adaptation of polystyrene patterning techniques. Experimental analysis of substrate-dependent effects on cell culture adhesion and organisation was extended to a non-cancerous cell line model. C2C12 mouse skeletal muscle cells were chosen for these investigations because of their ability to differentiate further, into myocytes or myofibrils. C2C12 myoblasts seeded on common cell culture substrates showed a notable morphology variation and extent of differentiation between cells grown on tissue culture polystyrene [TCPS] and polydimethylsiloxane [PDMS]. Myoblasts were plated on geometrically-patterned polystyrene and PDMS substrates. Significant alignment to grated pattern features was observed on both substrate types, before and after driven differentiation. Peeling artefacts of confluent tissue-like culture from PDMS surfaces which were observed were unreported previously in literature. The results reported in this thesis provide a foundation for potential research and commercial application for surface modification methods. The biomimetic topography provided by bioimprinted substrates can be used to identify and investigate cell activities, including for example the mechanisms of cell adhesion and separation in metastatic and invasive cancer research. Altering the material of the bioimprinted substrates may attune substrate topographies as scaffolds to direct specific stem cell differentiation for regenerative tissue engineering applications.

Exocytosis is a fundamental cellular process where membrane-bound secretory granules from within the cell fuse with the plasma membrane to form fusion pore openings through which they expel their contents. This mechanism occurs constitutively in all eukaryotic cells and is responsible for the regulation of numerous bodily functions. Despite intensive study on exocytosis the fusion pore is poorly understood. In this research micro-fabrication techniques were integrated with biology to facilitate the study of fusion pores from cells in the anterior pituitary using the atomic force microscope (AFM). In one method cells were chemically fixed to reveal a diverse range of pore morphologies, which were characterised according to generic descriptions and compared to those in literature. The various pore topographies potentially illustrates different fusion mechanisms or artifacts caused from the impact of chemicals and solvents in distorting dynamic cellular events. Studies were performed to investigate changes in fusion pores in response to stimuli along with techniques designed to image membrane topography with nanometre resolution. To circumvent some deficiencies in traditional chemical fixation methodologies, a Bioimprint replication process was designed to create molecular imprints of cells using imprinting and soft moulding techniques with photo and thermal activated elastomers. Motivation for the transfer of cellular ultrastructure was to enable the non-destructive analysis of cells using the AFM while avoiding the need for chemical fixation. Cell replicas produced accurate images of membrane topology and contained certain fusion pore types similar to those in chemically fixed cells. However, replicas were often dehydrated and overall experiments testing stimuli responses were inconclusive. In a preliminary investigation, a soft replication moulding technique using a PDMS-elastomer was tested on human endometrial cancer cells with the aim of highlighting malignant mutations. Finally, a Biochip comprised of a series of interdigitated microelectrodes was used to position single-cells within an array of cavities using positive and negative dielectrophoresis (DEP). Selective sites either between or on the electrode were exposed as cavities designed to trap and incubate pituitary and cancer cells for analysis by atomic force microscopy (AFMy). Results achieved trapping of pituitary and cancer cells within cavities and demonstrated that positive DEP could be used as a force to effectively position living cells. AFM images of replicas created from cells trapped within cavities illustrated the advantage of integrating the Biochip with Bioimprint for cellular analysis.