Stem cells have the ability to repair, replace or regenerate tissues. As a result their potential for regenerative medicine is vast. The processing of cells for therapeutic use and clinical diagnostics will rely on cell sorting steps to ensure a homogeneous population is obtained. Existing cell sorting technologies, either rely on the physical properties of cells, which tend to provide poor specificity' or require cells to be labelled with antibodies. Many of these techniques also tend to be expensive. Consequently there is a need for a fully synthetic, inexpensive, label-free separation system, capable of sorting cells with minimum manipulation. Initially, the generation of suitable substrates was investigated for high throughput polymer functionalisation in order to generate chemically heterogeneous surfaces through microarrays with the potential to induce differential cell adhesion. There was a need to develop immobilisation systems that were compatible with the high-throughput production of microarrays. The grafted-from and grafting-to methodologies were explored to find a suitable and versatile system, for high-throughput fabrication and immobilisation. The grafting-from approach relied on the generation of polymer brushes from a suitable low-fouling substrate. Despite the success of this technique, compatibility with high-throughput systems was limited. Therefore a grafting to approach was devised, in which specialist RAFT polymers could be immobilised to low fouling substrates using mid conditions, compatible with high-throughput systems. However the ability to produce a library of specialised polymers was limited. Consequently, a method was developed to immobilize thiol-functionalised materials to a low-fouling polymer substrate using thiol-ene click chemistry in a high-throughput format to fabricate immobilised microarrays. A heterogeneous population of cells derived from mouse embryoid bodies was subsequently seeded onto the arrays. Immunohistochemistry techniques were employed to track the differentiation of cells into different lineages. This technique allowed for the high-throughput quantification and identification of lineage specific cells by the means of automated fluorescence imaging and analysis. Lineage specific cell density was shown to vary according to material combination. Combinatorial chemistry was shown to be paramount in the adhesive selectivity of cells derived from mesoderm, endoderm and ectoderm lineages. Although glucose was shown to be the major component for each best-performing material, the addition of a minor component altered the surface chemistry and the specific adhesive properties for each material. In the future, successful materials are investigated further to generate structure-activity relationships. Ultimately we seek the generation of new surface-based devices that have the capacity to be fully synthetic, selective, inexpensive and disposable.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:659289 |
| Date | January 2013 |
| Creators | Simoes, Fabio |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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