Robust production of uniform human cerebral organoids from pluripotent stem cells

Abstract

Human cerebral organoid (hCO) models offer the opportunity to understand fundamental processes underlying human-specific cortical development and pathophysiology in an experimentally tractable system. Although diverse methods to generate brain organoids have been developed, a major challenge has been the production of organoids with reproducible cell type heterogeneity and macroscopic morphology. Here, we have directly addressed this problem by establishing a robust production pipeline to generate morphologically consistent hCOs and achieve a success rate of >80%. These hCOs include both a radial glial stem cell compartment and electrophysiologically competent mature neurons. Moreover, we show using immunofluorescence microscopy and single-cell profiling that individual organoids display reproducible cell type compositions that are conserved upon extended culture. We expect that application of this method will provide new insights into brain development and disease processes.

A matrigel-free method to generate matured human cerebral organoids using 3D-Printed microwell arrays

“…Previously, microwell platforms were used for the culturing of 3D spheroids, which often lack high-level structures. Three characteristics of microwells were believed critical for the formation of cell aggregates: spatial confinement for defined cell numbers [ 26 ], an anti-fouling smooth surface for non-adhesive cell contacts [ 17 ], and a concave curvature to facilitate spherical aggregation [ 21 , 31 , 35 , 41 ]. While the seeding cell density can help determine the former function, the latter two characteristics are mostly regulated through device fabrication and treatment.…”

“…Long-term culturing in Matrigel was also shown to lead to pre-mature organoids [ 14 ] and induce undesired neurite outgrowth [ 15 ]. Although synthetic extracellular matrix (ECM) substitutes were recently demonstrated [ 16 ], removing these materials (necessary for cell or imaging analysis) is still problematic because it unavoidably inflicts mechanical damage to the organoids [ 17 , 18 ]. Thus, finding alternative ECM-free methods continues to be one major topic of this field.…”

“…Microwell platforms [ 17 ] and other microfluidic 3D culturing platforms [ 19 , 20 ] have been successfully employed for the generation of spheroids [ [21] , [22] , [23] ], which are similar to organoids but lack high-level physiological features. When forming spheroid cultures, microwell platforms provide a geometrically-controlled micro-environment that defines spheroid biology (e.g., shape, size, and differentiation) [ [24] , [25] , [26] ].…”

“…Microwell platforms have been used in facilitating the rudimentary aggregation of embryoid bodies (EBs), which are spheroids of stem cells and the precursor of cerebral organoids [ 2 , 15 , 17 , 34 ]. Microwell platforms aided the initial self-organization of EBs, by providing a confined space (such as reverse-pyramid-shaped AggreWell [ 2 ] and curved surface [ 24 , 32 , 35 ]), enabling cell aggregation, and controlling initial cell densities [ 32 ].…”

“…Microwell platforms aided the initial self-organization of EBs, by providing a confined space (such as reverse-pyramid-shaped AggreWell [ 2 ] and curved surface [ 24 , 32 , 35 ]), enabling cell aggregation, and controlling initial cell densities [ 32 ]. Lately, microwell-derived EBs were further cultured to develop into organoids [ 17 , 34 ], however, still relying on Matrigel-embedding for modulating the organoid micro-environment [ 13 , 17 , 34 ]. In addition, most of the microwell generated spheroids (structureless aggregation) [ 36 , 37 ] or spheroid-like organoids (e.g.…”