Mass production of lumenogenic human embryoid bodies and functional cardiospheres using in-air-generated microcapsules

Organoids are engineered 3D miniature tissues that are defined by their organ-like structures, which drive novel fundamental understanding of human development and can revolutionize our healthcare system. However, current organoid generation methods associate with low production throughputs and poor control over size and function due to merging, which limits their clinical and industrial translation. Here, we present an innovative microfluidic platform for mass production of functional organoids. Specifically, triple-jet In Air microfluidics (IAMF) enables ultra-high throughput generation of hollow, thin-shelled, hydrogel microcapsules that can act as organoid forming bioreactors in a cytocompatible, oil-free, and size-controlled manner. IAMF microencapsulation of human pluripotent stem cells (hPSCs) allowed for long-term stable 3D suspension culturing and self-organizing hPSC spheroids with maintained pluripotency. Uniquely, IAMF microcapsules provided a lumenogenic microenvironment with near 100% efficient cavitation of hPSC spheroids resembling embryoid body's rosette-stage; autonomous self-organization with polarized cytoarchitecture was achieved without added growth factors. Upon chemical stimulation, hPSC-derived spheroids underwent cardiomyogenic differentiation effectively resulting in mass production of a homogeneous and functional (i.e., contracting) cardiospheres that were responsive to external electrical stimulation. In short, we report on an innovative method that enables efficient production of high-quality organized spheroids, embryoid bodies, and organoids in a cytocompatible, ultra-high-throughput, and clean manner, thereby driving clinical and industrial adaption of stem cell technology in application areas such as tissue engineering, drug testing, and cultured meat. Overall design: Comparative scRNA-seq analysis of encapsulated embryoid bodies and cardiospheres tissues and control (non-encapsulated) embryoid body tissues.