Development of a device for multi-modal mechanical manipulation of cells in 2D and 3D engineering environment

All organisms from bacteria to cells within the human body experience some form of mechanical stimuli. The biochemical response from mechanical stimuli is known as mechanotransduction. Cell manipulation devices provide an understanding of mechanotransduction and the various signalling mechanisms that take place. The objective of this Master’s thesis was to develop a device for multi-modal mechanical manipulation of cells in 2D and 3D environments. The device is to mimic the stress conditions or the mechanical environment of the cells in vitro. The mechanical cell loading device will be used to perform cellular mechanical experiments to assist in other future biophysical research and investigate the mechanics of cells under various degrees of tension, compression and shear so that a better understanding of mechanotransduction can be obtained. Cells are seeded in a biocompatible medium and their force response is observed. The incorporation of tension, compression and shear stress in a single device constitutes the uniqueness of this designed device. A cell manipulator device was designed and assembled with different modular attachments for the various kinds of stress loading. The dimensions of the device were selected in a manner to enable the device to be mountable on a microscope for live cell imaging. The Carl Zeiss LSM510 Confocal Microscope was the microscope available for the experimentation. In this project, live cell imaging is only possible with tensile strain. Thus, the tension system was the predominant focus. Live cell imaging during tension provides accurate information about cellular morphology. Three different types of PDMS membranes were designed, manufactured and tested by applying a tensile load from the designed device. The three types of PDMS membranes produced were: 20 mm x 20 mm, 20 mm x 20 mm with 1mm thickness dividers (dividers divided the PDMS membrane into 4 even sized quadrants), and 10 mm x 10 mm. Strain characterisation of the three types of PDMS membrane was performed. The PDMS membranes are marked with ink from a permanent marker which serves as a frame of reference for strain measurement. Using the permanent marker, dots were marked in grid format. The PDMS membranes were subjected to tensile stress from the designed device under a confocal microscope. Length deformation of the markers along the stretch axis was measured and recorded during the practical experimentation. Using FEA software, FEA models of each type of PDMS membrane was simulated. The purpose of the FEA models is to facilitate the future studies of researchers. FEA simulations provide feedback to guide actual cellular experimentation for researchers. The FEA models of the various types of PDMS membranes were validated against the practical experimentation of strain characterisation. From the analysis and discussion of the results of FEA and practical experimentation, the designed device satisfies the objectives of this project. The device was most successful with the 20 mm x 20 mm PDMS membrane type since it showed close correlation to the ideal strain output. FEA simulation of the 20 mm x 20 mm PDMS membrane also showed close correlation to the experimental results. But, in the instance of the 10 mm x 10 mm PDMS membrane, experimental results of the strain output did not correspond with the user strain due to the clamping mechanism unable to grapple PDMS membrane appropriately. Thus, validation of the FEA 10 mm x 10 mm PDMS membrane was not successful

DEVELOPMENT OF A THERAPEUTIC MODEL OF EARLY LIVER CANCER USING CROCIN-COATED MAGNETITE NANOPARTICLES

Hepatocellular carcinoma is one of the most common health problems that is difficult to treat. As a result of the side effects frequently experienced with conventional cancer treatments, there has been a growing interest to develop controlled drug delivery system that can reduce the mortality rate of liver cancer patients and un-harm healthy tissues. Magnetite nanoparticles are potentially important in hepatocellular carcinoma treatment, since they can be used as delivery system. Pure and coated magnetite nanoparticles were synthesized via modified co-precipitation method in air at low temperature. Various reaction parameters and coating materials have been investigated and characterized. Among these parameters and coating materials, 1.0 % of dextran was selected as an optimum coating for nanoparticles using a slow feeding rate for the Fe2+/Fe3+ reactants, maintaining the stirring and soaking temperatures at 60ºC. After that dextran-coated magnetite nanoparticles were bound to crocin, a pharmacologically active component of saffron, via cross-linker. Crocin alone has shown anti-cancer activity in different in vitro and in vivo settings by several studies. The aim of this study was to synthesize dextran-coated magnetite nanoparticles containing crocin with a higher therapeutic index for hepatocellular carcinoma treatment. The nanoparticles with crocin were tested in vitro and in vivo for their anti-cancer effects as compared to free crocin. HepG2 cells treated with crocin-dextran-coated magnetite nanoparticles showed a decrease in cell proliferation compared to control (non-treated cells) or to those treated with free crocin or dextran-coated nanoparticles. The anti-cancer activity of crocin-dextran-coated nanoparticles was also evaluated in Balb/c mice. These mice were injected with carcinogenic agent, diethylnitrosamine. Histological examination revealed several precancerous changes. The immunohistochemical analysis using antibodies indication of cell proliferation (Ki-67), apoptosis (M30-Cytodeath and Bcl-2), inflammation (cyclooxygenase-2) and angiogenesis (vascular endothelial growth factor), indicated that magnetite nanoparticles conjugated with dextran plus crocin does indeed improve its anti-tumorigenic activity over free crocin. These results provide the basis for designing new modalities for treatment of liver cancer which could hopefully reduce its high mortality rate

Photodynamic Therapy 2021

Photodynamic therapy (PDT) is a light-based photochemistry process. The illumination of a photoactivatable molecule (also called photosensitizer) with visible or near infrared light produces reactive oxygen toxic species to destroy tumor cells. This treatment modality leads to highly targeted actions, because reactive oxygen species are produced only where light is applied. Light is not harmful, nor is the photoactivable molecule. Only the combination of three elements (photosensitizer, oxygen, and light) is required to induce photo-oxidation reactions. PDT has proven to be a promising modality in many medical applications including cutaneous condition, infectious diseases, and various cancers at different stages. This book is a reprint of the Special Issue "Photodynamic Therapy 2021" that was published in Pharmaceuticals

The role of bone morphogenetic protein signalling in the control of skin repair after wounding. Cellular and molecular mechanisms of cutaneous wound healing mediated by bone morphogenetic proteins and their antagonist Noggin.

Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) coordinate tissue development and postnatal remodelling by regulating proliferation, differentiation and apoptosis. However, their role in wound healing remains unclear. To study this, transgenic mice overexpressing Smad1 (K14-caSmad1) or the BMP antagonist Noggin (K14-Noggin) were utilised, together with human and mouse ex vivo wound healing models and in vitro keratinocyte culture. In wild-type mice, transcripts for Bmpr-1A, Bmpr-II, Bmp ligands and Smad proteins were decreased following tissue injury, whilst Bmpr-1B expression was up-regulated. Furthermore, immunohistochemistry revealed a down-regulation of BMPR-1A in hair follicles adjacent to the wound in murine skin, whilst in murine and human wounds, BMPR-1B and phospho-Smad-1/5/8 expression was pronounced in the wound epithelial tongue. K14-caSmad1 mice displayed retarded wound healing, associated with reduced keratinocyte proliferation and increased apoptosis, whilst K14-Noggin mice exhibited accelerated wound healing. Furthermore, microarray analysis of K14-caSmad1 epidermis revealed decreased expression of distinct cytoskeletal and cell motility-associated genes including wound-associated keratins (Krt16, Krt17) and Myo5a versus controls. Human and mouse keratinocyte proliferation and migration were suppressed by BMP-4/7 both in vitro and ex vivo, whilst they were stimulated by Noggin. Additionally, K14-caSmad1 keratinocytes showed retarded migration compared to controls when studied in vitro. Furthermore, Bmpr-1B silencing accelerated migration and was associated with increased expression of Krt16, Krt17 and Myo5a versus controls. Thus, this study demonstrates that BMPs inhibit proliferation, migration and cytoskeletal re-organization in epidermal keratinocytes during wound healing, and raises a possibility that BMP antagonists may be used for the future management of chronic wounds

Metastatic cell responses to anticancer therapy: The possible role of cancer stem cells

Metastasis is a multistep process, by which cancer cells dissociate from the initial site and travel to form new secondary tumours at distant sites. This process has been reported to require epithelial-mesenchymal transition (EMT), by which the epithelial cancer cells convert to a mesenchymal form, associated with increasing levels of vimentin and decrease in E-cadherin markers. Cancer metastasis is also associated with aberrations in cells’ glycosylation such as Tn antigen which can be detected by Helix pomatia agglutinin (HPA) lectin extract from Helix pomatia snails. Radiotherapy could have an effect on cancer metastasis; by stimulating regulation signalling including ROS, TGF-β, E-cadherin and vimentin. This signalling can promote cancer cells to change from the epithelial to mesenchymal form. The cells behaviour could be mediated by the production of extracellular vesicles/exosomes from irradiated cells, which may have an impact on unirradiated cells. To date, the role of direct and bystander effect of ionising irradiation on the cancer cells glycosylation, EMT markers and invasiveness has not yet been investigated. Therefore, a study has been established to determine the direct effect, and if the progeny of cancer cells that survive a therapeutic dose of X-radiation show differences in aberrant glycosylation associated with metastasis, EMT markers and their ability to invade compared to unirradiated cells. The study also aims to investigate the bystander effect of extracellular vesicle/exosomes in the invasion of breast cancer cells. The molecular mechanisms that could be involved in the invasion of breast cancer cells following irradiation or exosome transfer were also investigated. In this study, both cells directly exposed to a therapeutic dose of ionising radiation and unirradiated recipient cells treated with exosomes from irradiated cells showed an increase in vimentin immunopositivity, HPA positivity, a decrease in the E-cadherin immunopositivity and a significant increase in the invasive capacity of the cells. The study also showed that breast cancer cells treated with a single dose of ionising irradiation induce invasion of breast cancer cells in vitro. The findings were further confirmed using qPCR and western blot for examination of selected genes and proteins that have an effect on the invasion of cancer cells following direct ionising irradiation or bystander effect of exosomes and may have an impact on cancer metastasis. The study also showed that exosomes isolated from irradiated cells carried a high quantity of TGF-β protein and specific miRNA which have an impact on the invasion of cancer cells. This study concludes that single therapeutic dose of ionising irradiation induces invasion of cancer cell in vitro. In addition, exosomes and its compounds that were isolated from irradiated cell condition media can promote invasion of breast cancer cells, and that could have an implication on cancer therapy in the future

Biocompatible tumour implant systems: towards an integrated biophotonic system

There is a need to perform comprehensive cell biology studies transferable across culture platforms using innovative cellular models. The higher purpose is to bridge the gap between in vitro cell culture and in vivo models. In this thesis a significant advance is presented in the embedding of an innovative optical biophotonic capability for the dynamic interrogation and single cell tracking of human osteosarcoma cells encapsulated in the hollow fiber (HF) platform. Two approaches have been implemented: quantum dot (QD) nanoparticles providing proliferative and cell cycle readouts and an in-fiber light illumination providing global features of particle and cell density. An in vitro HF encapsulation model was developed and characterised against standard two-dimensional tissue culture (TC) using the human osteosarcoma U-2 OS cell line expressing a cell cycle fluorescent reporter (cyclin Bl-GFP). Analysis of the packing and orientation of cells in the HF revealed that they grow like an anchorage dependent adherent layer. Overall cells in the fiber displayed a slower cell cycle traverse and a differential sensitivity to clinically relevant doses of the anticancer mitosis-inhibiting agent Taxol compared to cells under normal TC conditions. Comprehensive gene profiling, with bioinformatics and ontology network analysis, showed that the HF cells presented high steroid related but low differentiation gene expression. Specific biomarkers were indentified, and it is suggested that the HF model displays features that are closer to an in vivo tumour. A flow cytometry cell-tracking approach using QD labelling was validated and applied to the HF model for the first time. This represents an "embedded" biophotonic system where the QD sensors are integrated directly into the seeded cell population and then redistributed through the daughter cells, thus reflecting patterns of lineage expansion. This provides sub-population parameterized information on cell-cell heterogeneity and cell division. A biophotonic HF prototype comprising the integration of direct coupled-light excitation in the HF was conceived, this revealed the potential and limitations to detect die presence of cells inside the HF lumen by analysing light attenuation changes. Finally a "systems cytometry" acquisition concept has been proposed, comprising the use of embedded engineered nanoparticles as single cell "nano-memory" biophotonic intracellular probes