High‐Yield Convenient Mass Production of High‐Quality Homogenous Human Induced Pluripotent Stem Cell Spheroids under Rho‐Associated Kinase Inhibitor‐Free 3D Culture Enabled by Micropatterning and Cold‐Triggered Chemical‐Free Cell Detachment

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Human induced pluripotent stem cells (hiPSCs) show great promise for personalized cell-based medicine, as they can be derived from easily accessible somatic cells and differentiated into all three germ layers without ethical concerns. This requires mass production of hiPSCs in 3D. However, contemporary methods for 3D culture result in hiPSC spheroids with significant size heterogeneity that is undesired for controlled differentiation and require the use of a high concentration of Rho-associated kinase inhibitor (RI) to improve the cell viability. Unfortunately, a high concentration of RI causes uncontrolled spontaneous differentiation. To address these challenges, a cold-responsive micropatterned dish (crMPD) is developed by spin-coating a thin layer of cold-responsive polymer on a cell culture dish and further microcontact printing cell attachment micropatterns on top of the coating layer. The hiPSCs attach and proliferate exclusively within the micropatterned areas to form a large number of uniform hiPSC colonies that can be detached as a whole by putting the crMPD on ice for ≈5-15 min. Under 3D culture without RI, the colonies can quickly self-assemble into homogeneous hiPSC spheroids with high viability, yield, and pluripotency. This ingeniouscrMPD technology may be invaluable to facilitate widespread application of hiPSCs in research and personalized medicine.