Developing a novel spheroid-on-chip microfluidic device for investigations into metastasis

Collins, Thomas Charles

Abstract

One in two people born after 1960 will develop cancer, with 90% of all cancer deaths arising from metastasis. Conventional 2D in vitro metastasis models do not fully replicate tumour complexity. In vivo models can address tumour complexity, but do not fully represent human tumour biology. Multicellular spheroids are widely used 3D models of cancer. Spheroids contain internal zonal differentiation of oxygen, metabolite, and nutrient gradients, associated with regions of proliferative, quiescent, and necrotic cells. Current in vitro static spheroid methodologies do not recapitulate factors of cellular spread including continuous flow or shear stress. Therefore, better 3D in vitro models to investigate metastasis represent an area of unmet need. This research aims to develop spheroid-on-chip models, providing a novel strategy to investigate cancer spread. The microfluidic devices used in this study feature weirs for spheroid inclusion, and a borosilicate base coverslip for optical clarity. The devices feature an access port allowing direct access to the microwell. Spheroids derived from established cancer cell lines, MCF7 and U-87 MG, were formed off-chip and incorporated into the device by pipetting, before being perfused with complete media at 3 μL min-1 for 72 h. Cell viability was assessed in effluent, using the CytoTox Glo assay, demonstrating spheroid viability is robustly maintained on-chip. In situ analysis of cell viability, through FDA/PI live/dead staining indicated an increased proportion of viable cells and decreased dead cells on-chip compared to off-chip. VEGF (Vascular Endothelial Growth Factor) ELISA showed that VEGF secretion, as evaluated by its presence in conditioned media, was comparable across all testing conditions. The chip model has been further developed to allow spheroids to be embedded in ECM (extracellular matrix)-like matrices. VEGF ELISA and IL-6 ELISA showed that both VEGF and IL-6 secretion, as evaluated by its presence in conditioned media, was comparable across all testing conditions. ELISA also showed that IL-6 and VEGF was increased in the on-chip models within hydrogel conditions. The device has also allowed the direct imaging of spheroids on chip over 72 h. The analysis of U-87 MG spheroids on chip showed that invasion through Matrigel was comparable to the off-chip static models. Whilst migration on chip, through collagen analysis, was increased over the off-chip counterparts. The work shown offers a novel insight into cancer metastasis; on a more replicative model than the current conventional in vitro techniques.

Citation

Collins, T. C. Developing a novel spheroid-on-chip microfluidic device for investigations into metastasis. (Thesis). University of Hull. https://hull-repository.worktribe.com/output/4221851

• Thesis Type: Thesis
• Deposit Date: Oct 9, 2019
• Publicly Available Date: Feb 23, 2023
• Keywords: Biomedical sciences
• Public URL: https://hull-repository.worktribe.com/output/4221851
• Additional Information: School of Biological, Biomedical and Environmental Sciences, The University of Hull
• Award Date: May 1, 2019