Microfluidic, Stem Cell, Aging
Within in vivo tissue niches, stem cell fate is exquisitely controlled by a combination of biochemical, physico-chemical and mechano-structural factors. Any perturbation in this tightly controlled microenvironment can lead to cellular dysfunction. The compositional and temporal nature of the "niche" within ex vivo environs must thus be similarly controlled if we wish to invoke explicit control over stem cell behaviours so that we may manipulate stem cell fate choices (expansion, senescence delay, differentiation etc.) during ex vivo culture. Microfluidic bioreactor arrays (MBAs), as recently invented within the Cooper-White laboratory, permit the investigation of cellular responses to hundreds of exogenously-provided factor combinations, whilst also permitting one to study the impacts of secreted (endogenously-provided) soluble factors (paracrine signalling) at the same time. Whilst this device platform allows the rapid screening of a variety of soluble factors (growth, maintenance and differentiation factors), other important microenvironmental variables, such as dissolved oxygen concentration, pH and media exchange rates, remain unmeasured and uncontrolled, reducing the ability of the device to truly optimise stem cell culture outcomes and transfer such optima to larger scale bioreactors. Real time sampling and isolation of cellular material is also restricted. This project aims to firstly address these deficiencies by a) integrating real-time measurements of pH, dissolved oxygen concentration and flow rate into a microfluidic bioreactor device platform; and b) permitting the isolation of each nanolitre culture environment in order to extract cells and cellular secretions from defined microenvironment for further molecular analysis, such as gene and protein expression. Once this next generation MBA platform has been realised, it will be used to explore microenvironmental conditions that actively inhibit or delay mesenchymal stem cell (MSC) senescence during extended ex vivo expansion, a significant roadblock to their effective use in tissue engineering and regenerative medicine applications.