Three-dimensional microfluidics with dynamic fluidic perturbation promotes viability and uniformity of human cerebral organoids.

Human cerebral organoids (COs), generated from stem cells, are emerging animal alternatives for understanding brain development and neurodegeneration diseases. Long-term growth of COs is currently hindered by the limitation of efficient oxygen infiltration and continuous nutrient supply, leading to general inner hypoxia and cell death at the core region of the organoids. Here, we developed a three-dimensional (3D) microfluidic platform with dynamic fluidic perturbation and oxygen supply. We demonstrated COs cultured in the 3D microfluidic system grew continuously for over 50 days without cell death at the core region. Increased cell proliferation and enhanced cell differentiation were also observed and verified with immunofluorescence staining, proteomics and metabolomics. Time-lapse proteomics from 7 consecutive acquisitions between day 4 and day 30 identified 546 proteins differently expressed accompanying COs growth, which were mainly relevant to nervous system development, in utero embryonic development, brain development and neuron migration. Our 3D microfluidic platform provides potential utility for culturing high-homogeneous human organoids.