Islet transplantation holds vast potential as a treatment for type 1 diabetes mellitus and provides recipients with short term insulin independence. A major limitation of this treatment is the lack of donor beta (β) cells available for transplantation. Significant progress has been made with stem cell differentiation protocols; current methods have generated cell populations which possess a functioning β cell phenotype. However, these cells are not suitable for clinical transplantation. Two dimensional cell culture systems do not accurately mimic the complexity of the in vivo pancreatic environment, reducing the effectiveness of current β-cell differentiation protocols. The paradigm shift into three dimensional tissue culture provides an attractive area of investigation, and the use of three dimensional culture methods has improved growth in a variety of cell types ex vivo. The mass culture of β cell analogues on a 3D biomimetic environment is now necessary, and may offer a new platform on which an alternative source of transplantable cell populations can be differentiated and cultured successfully. This thesis aims to develop and condition BioVyon™, a high density polyethylene (HDPE) based biomaterial, for use as a mass β cell cultivation system In order to achieve this, a number of objectives will need to be met: (I) Complete characterisation and assessment of all properties of Biovyon™ that have a direct influence on cell culture, (ii) modification of the BioVyon™ surface chemistry to promote cell adhesion and growth, (iii) absorbance of proteins to mimic the pancreatic environment and aid proliferation of the Min-6 cell line, (iv) assessment of the Min-6 cell line phenotype after extended period of culture on the modified BioVyon ™ environment. Scanning electron microscopy and Atomic Force Microscopy were used to characterise the surface of the HDPE material post plasma etching. Advancing and receding (ARCA) and Fourier Transform Infrared Spectroscopy were used to analyse the elemental changes to the polymer surface. The HDPE biomaterial was conditioned using plasma etching, subsequent adhesion and growth of Min-6 cells was quantified using a lactate dehydrogenase assay. Min-6 populations were seeded at a density of lxl0/6i on BioVyon™ and tissue culture plastic of comparable surface area, and analysed after extended periods of growth. An insulin EUSA was used to quantify insulin released by populations of β cells at different time points witin the BioVyon™ in response to fluctuating glucose concentrations. The results obtained in this thesis indicate that BioVyon™ offers an appropriate structural environment for cell culture. Pore size and frit dimensions allow for cell infiltration and the effective diffusion of oxygen and nutrients. Plasma etching incorporated oxygen groups and a novel surface topography that improved cell adhesion and growth. β-cell phenotype was protected and sustained in cell populations cultured within the BioVyon™ environment. In conclusion, BioVyon™ can be conditioned to function as an effective 30 cell culture system. Modification of the surface chemistry has enabled BioVyonTl • to harbour and sustain large populations of the Min-6 cell line. The protected β-cell phenotype in the Min-6 populations suggests BioVyon™ could hold potential as a stem cell culture and differentiation platform.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:608311 |
| Date | January 2013 |
| Creators | Bockhart, James David |
| Publisher | University of Brighton |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://research.brighton.ac.uk/en/studentTheses/06e474c1-fbd9-49d7-89ef-55c4e571ee42 |
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