Long-Term Retinal Differentiation of Human Induced Pluripotent Stem Cells in a Continuously Perfused Microfluidic Culture Device.

Understanding how microenvironmental cues influence cellular behavior will enable development of efficient and robust pluripotent stem cell differentiation protocols. Unlike traditional cell culture dishes, microfluidic bioreactors can provide stable microenvironmental conditions by continuous medium perfusion at a controlled rate. The aim of this study is to investigate whether a microfluidic culture device could be used as a perfused platform for long-term cell culture processes such as the retinal differentiation of human induced pluripotent stem cells. The perfusion flow rate is established based on the degradation and consumption of growth factors (DKK-1, Noggin, IGF-1, and bFGF) and utilizing the Péclet number. The device's performance analyzed by qRT-PCR show improvements compared to the well-plate control as characterized by significantly higher expression of the markers Pax6, Chx10, and Crx on Day 5, Nrl on day 10, Crx, and Rhodopsin on day 21. Optimization of perfusion rate is an important operating variable in development of robust processes for differentiation cultures. Result demonstrates convective delivery of nutrients via perfusion has a significant impact upon the expression of key retinal markers. This study is the first continuously perfused long-term (21 days) retinal differentiation of hiPSCs in a microfluidic device.

Transfection in perfused microfluidic cell culture devices: A case study.

Automated microfluidic devices are a promising route towards a point-of-care autologous cell therapy. The initial steps of induced pluripotent stem cell (iPSC) derivation involve transfection and long term cell culture. Integration of these steps would help reduce the cost and footprint of micro-scale devices with applications in cell reprogramming or gene correction. Current examples of transfection integration focus on maximising efficiency rather than viable long-term culture. Here we look for whole process compatibility by integrating automated transfection with a perfused microfluidic device designed for homogeneous culture conditions. The injection process was characterised using fluorescein to establish a LabVIEW-based routine for user-defined automation. Proof-of-concept is demonstrated by chemically transfecting a GFP plasmid into mouse embryonic stem cells (mESCs). Cells transfected in the device showed an improvement in efficiency (34%, n = 3) compared with standard protocols (17.2%, n = 3). This represents a first step towards microfluidic processing systems for cell reprogramming or gene therapy.