Towards Rapid Label-free Enrichment of Specific Stem Cell Populations for Autologous Cell Therapies

Autologous mesenchymal stem cell (MSC) therapies have huge potential in addressing clinical challenges for otherwise intractable diseases. Label-free, intra-operative separation and enrichment of MSC subpopulations would provide a step change in delivery of such therapies. The long term goal of this research is to use binding proteins to provide a surface with switchable affinity, coupled with microfluidics to selectively bind and subsequently collect released cells. The specific aim of this thesis was to take the first steps towards achieving this goal, by identifying the most suitable binding proteins for cell capture and release in a prototype device and determining the feasibility of cell enrichment from complex clinical samples such as bone marrow aspirate. A prototype device was developed exploiting the cell surface marker CD271 to select for MSCs. Affimer binding proteins and a commercially available antibody were investigated for specific cell capture and release. Specificity for CD271+ cells was demonstrated via flow cytometry using two different cell types. CD271 binding proteins were immobilised to a low-fouling substrate in a microfluidic channel and known mixtures of the two cell populations used to demonstrate specific cell capture. Increased flow rates allowed for bound cells to be released, collected and analysed, providing evidence that cells remained viable and minimally manipulated after enrichment. Clinical samples of bone marrow aspirate were then used in the same way and the results compared to gold standard methods of cell sorting. Results showed that the percentage of CD271+ cells selected from bone marrow mononuclear cell populations using the prototype device was similar to results obtained using established cell sorting methodologies. This work demonstrated that affinity capture via antibody technology, together with a surface designed to provide a controlled release mechanism, offers a high-throughput, minimally manipulative approach to select and enrich MSC populations for therapeutic applications