In this thesis, effects of stable concentration of key growth factors on retinal differentiation of human induced pluripotent stem cells (hiPSCs) were investigated in a microfluidic culture device (MFCD). In vitro culture dishes such as flasks and well-plates lack microenvironmental controls required for maintaining stable culture conditions. Unlike cell culture dishes, microfluidic devices provide greater microenvironmental controls resulted from shorter characteristic length and use of laminar flow. It is hypothesised that using the MFCD, a flow rate can be found which keeps key growth factors in retinal differentiation culture in steady-state. Subsequently, steady-state concentration of growth factors would result in upregulation of retinal progenitor (Pax6, Lhx2, Six6 and VSX2/Chx10) and precursor markers (Crx and Nrl). Initially, degradation and consumption of growth factors (DKK-1, Noggin, IGF-1 and bFGF) used in retinal differentiation were studied to establish an order of importance. These findings along with dimensionless ratios, Péclet and Damköhler numbers, were utilised to establish a perfusion rate of 130 μL/h. At this flow rate growth factors DKK1 and Noggin were delivered to the cells in steady-state conditions. A second perfusion culture with the same media exchange rate as the static culture at flow rate of 5.2 μL/h was added to act as a second control. Perfusion cultures were performed for 5, 10, and 21 days (n=3). The MFCD with higher flow rate showed significantly higher expression of markers Crx (p<0.05) and Rhodopsin (compared to lower MFCD, p<0.05) on day 21. The MFCD with lower flow rate, showed significantly higher expression of Pax6 (p<0.05), Vsx2/Chx10 (p<0.01) and Crx (p<0.05) on day 5, Nrl (p<0.01) on day 10 and Six6 (p<0.05) on day 21. This was the first continuously perfused long-term (21 days) retinal differentiation of hiPSCs in a microfluidic device, and results illustrated the importance of steady-state conditions in stem cell bioprocessing
