Advanced migration assays for studying chemotaxis of slow moving cells

Abstract

Chemotaxis of slow moving cells plays a crucial role in numerous physiological and pathophysiological processes; including tumour metastasis, development, and wound healing. Therefore, a comprehensive understanding of chemotactic behaviour, and the characterisation of extracellular signals and cues that prompt directed cell migration is highly desirable. The state-of-the-art chemotaxis assays are in general designed either with respect to the migration characteristics of fast moving cells (such as cells of the immune system), or focus on an in-depth investigation of chemotactic behaviour, requiring a time-demanding and labour-intensive analysis of individual cell trajectories. Such approach poses a limitation for experiments challenged by an increased number of samples; e.g., screenings for chemoattractants, or molecules and genetic mutations with the potential to inhibit or promote the chemotactic effect. <br /> Therefore, the first aim of this thesis was to develop a novel chemotaxis assay suitable for slow moving cells (e.g., keratinocytes, cancer cells), which would enable fast and effortless evaluation of the chemotactic response. For that purpose, advanced stimuli-responsive materials and micro-patterning methods were applied, and a chemotaxis chamber with hydrogel migration arena was established. The assay employs a gradient generator that maintains a well-defined long-term stable chemical gradient, essential for the investigation of chemotaxis in slow moving cells. The design of the assay enables evaluation of the chemotactic effect from the end-point state of the experiment. This method substantially facilitates the analysis, providing for an increased experimental throughput. <br /> Characterisation and quantification of the chemotactic behaviour of whole cell populations provide the fundamental information on the chemotaxis-inducing stimuli and the relevant response, which is often cell-type specific. However, to get a complete picture of cell behaviour during chemotaxis, further detailed investigations of single cells in conditions close to their physiological environment is required. Therefore, in another part of the thesis, the focus was set on establishment of experimental procedures for studying the migratory behaviour of single chemotaxing cells in long-term stable gradients with advanced imaging techniques, such as traction force microscopy, and light-sheet fluorescence microscopy. <br /> Finally, the novel tool was employed to investigate the chemotaxis of primary keratinocytes, slow moving cells that play a central role in re-epithelialisation of wounds. For the first time, the normal human epithelial keratinocytes (nHEK) were exposed to chemical gradients of several growth factors over long time-periods. The chemotactic response to a range of varying chemical conditions was evaluated quantitatively with the end-point assay. From the tested substances, the epithelial growth factor (EGF) and transforming growth factor α (TGFα) were most potent in inducing the directed migration of nHEK cells. The identification and quantification of the parameters that prompt keratinocyte chemotaxis made it possible to establish a model for further investigations of gradient sensing and directed migration of epithelial cells, aiming for new therapeutic strategies to promote wound repair.