Tissue geometry drives deterministic organoid patterning.

Epithelial organoids are stem cell­derived tissues that approximate aspects of real organs, and thus they have potential as powerful tools in basic and translational research. By definition, they self-organize, but the structures formed are often heterogeneous and irreproducible, which limits their use in the lab and clinic. We describe methodologies for spatially and temporally controlling organoid formation, thereby rendering a stochastic process more deterministic. Bioengineered stem cell microenvironments are used to specify the initial geometry of intestinal organoids, which in turn controls their patterning and crypt formation. We leveraged the reproducibility and predictability of the culture to identify the underlying mechanisms of epithelial patterning, which may contribute to reinforcing intestinal regionalization in vivo. By controlling organoid culture, we demonstrate how these structures can be used to answer questions not readily addressable with the standard, more variable, organoid models.

Hydrogel-based milliwell arrays for standardized and scalable retinal organoid cultures.

The development of improved methods to culture retinal organoids is relevant for the investigation of mechanisms of retinal development under pathophysiological conditions, for screening of neuroprotective compounds, and for providing a cellular source for clinical transplantation. We report a tissue-engineering approach to accelerate and standardize the production of retinal organoids by culturing mouse embryonic stem cells (mESC) in optimal physico-chemical microenvironments. Arrayed round-bottom milliwells composed of biomimetic hydrogels, combined with an optimized medium formulation, promoted the rapid generation of retina-like tissue from mESC aggregates in a highly efficient and stereotypical manner: ∼93% of the aggregates contained retinal organoid structures. 26 day-old retinal organoids were composed of ∼80% of photoreceptors, of which ∼22% are GNAT2-positive cones, an important and rare sensory cell type that is difficult to study in rodent models. The compartmentalization of retinal organoids into predefined locations on a two-dimensional array not only allowed us to derive almost all aggregates into retinal organoids, but also to reliably capture the dynamics of individual organoids, an advantageous requirement for high-throughput experimentation. Our improved retinal organoid culture system should be useful for applications that require scalability and single-organoid traceability.