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The Impact of substrate texture on cell behavior
Traditionally, cell studies have focused mainly on the effects of biochemical signals on cellular behaviour; however, there is a growing interest in investigating the effects of physical cues on the behaviour of the cells. Development of fabrication techniques has allowed researchers to design topographical cues at the micro and nanometer scale. In this study, we fabricated fibronectin stripes, which mimic some of the frequent patterns that cells are exposed to in their natural microenvironment, and studied the effects of these patterns on 3T3 fibroblasts and human mesenchymal stem cells which are of significant importance in the field of regenerative medicine and tissue engineering. Our results show that cells align and elongate in the direction of the patterns and the morphological features of cell, nucleus and architecture of force-bearing stress fibers of the actin cytoskeleton are all highly dependent on the stripe width. 3D confocal measurements showed that the cell thickness and nuclear shape is regulated through the different arrangement of perinuclear actin stress fibers depending on the stripe sizes. We also observed that when the size of stripes (5 µm) is significantly smaller than the size of cell nuclei (subnuclear stripes), cells exhibit 3 different morphological responses to the patterns. Minimal confinement, branching on the stripes and elongation on a single stripe. The majority of cells and nuclei align themselves with the subnuclear stripes; however, a substantial number of hMSCs nuclei are elongated perpendicular to the direction of the pattern, a finding which we explained using a geometrical model based on cell size. The behaviour of 3T3 cell lines and the hMSCs were compared for the different types of pattern.
We designed setups that allow us to perform Raman microspectroscopy both in 2D and 3D topographical patterns in order to be able to study their morpho-chemical effects on cells. We also genetically modified 3T3 fibroblasts to express fluorescent-tagged proteins that visualize the actin cytoskeleton and the nucleus which allow us to perform live cell imaging to study the dynamics of cell behaviour on topographical patterns.Peterhouse College research studentship,
Cavendish laboratory research scholarship,
Cambridge Trust- IDB 3 years full PhD scholarshi
Cell death dynamics monitoring using Raman micro-spectroscopy
Biopharmaceuticals play a crucial role in curing diseases like Cancer and diabetes. Bioreactors are the heart of the industry. Cell losses due to cell death such as apoptosis and necrosis in the bioreactor decreases production efficiency and subsequently increases the cost of production. Furthermore, the study of apoptosis and necrosis cell death mechanisms has a great scientific and clinical importance in cancer therapy. In this project, Raman micro-spectroscopy is used to study apoptosis and necrosis in Chinese’s Hamster Ovary (CHO) cells that are one the main host cell lines used in the production of biopharmaceuticals. Apoptosis and Necrosis were induced in CHO cells using camptothecin and oxygen and glucose deprivation. The changes in the chemical composition of these enriched apoptotic and necrotic cell cultures were then analyzed using Raman spectroscopy which revealed novel biological concepts of the cell death process. Moreover, highly distinguishing Raman characteristics were identified for each death mode. These observations made by Raman spectroscopy were confirmed using a broad range of conventional and advanced biological assays in the field ranging from FACS analysis and fluorescent dyes to fluorescence microscopy. Studying Raman Spectra gave a clear image about DNA, RNA and Protein level changes during the process of apoptosis and necrosis in CHO cells. Using Principle Components Analysis (PCR) enabled viable, necrotic, early and late apoptotic populations to be clearly distinguished. This technology may provide the basis for the development of a non-invasive probe to monitor and predict cell death in bioreactor cultures in real-time and possibly allow cultures to avoid entering the cell death phase. In addition, the vast majority of cancer treatment methods involve cell death and apoptosis and, therefore, improving our knowledge about the biology of cell death will help support and advance research and treatment in this area.Science, Faculty ofPhysics and Astronomy, Department ofGraduat