Abstract
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.
Highlights
Understanding how microenvironmental cues influence cellular behavior will crucial when it comes to increasing enable development of efficient and robust pluripotent stem cell differentiation protocols
The modular design of the Microfluidic Culture Device (MFCD) was comprised of a gas permeable lid made from poly(dimethylsiloxane) (PDMS) and a lid holder from polycarbonate (PC), two interconnects made of PC, a top frame (PC), bottom frame and reinforcement brackets made from Aluminium (Al), a gasket and a microfluidic chip made from PDMS, and a tissue-culture polystyrene (TC-PS) slide as a culture surface, mirroring the same culture surface as conventional cell culture dishes
The lid design meant that EBs could be directly seeded into the culture chamber, avoiding the flow through seeding and/or specialized arrangements for in-situ EB formation. human induced pluripotent stem cells (hiPSCs) could be aggregated into EBs using standard techniques and directly pipetted into the cell culture chamber
Summary
Understanding how microenvironmental cues influence cellular behavior will crucial when it comes to increasing enable development of efficient and robust pluripotent stem cell differentiation protocols. The device’s performance analyzed by qRT-PCR show improvements compared to the well-plate control as characterhuman embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have been developed in traditional culture formats such as T-flasks and well-plates. These formats lack the microized by significantly higher expression of the markers Pax, Chx, and Crx on environmental controls required to main-. This study is the first continuously perfused long-term (21 days) retinal ronment present during embryonic develdifferentiation of hiPSCs in a microfluidic device
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