Title:
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Functionalised polymer surfaces for the selective attachment and sorting of mammalian cells
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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.
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