Efficient Generation of Skin Organoids from Pluripotent Cells via Defined Extracellular Matrix Cues and Morphogen Gradients in a Spindle‐Shaped Microfluidic Device

Abstract Pluripotent stem cell‐derived skin organoids (PSOs) emerge as a developmental skin model that is self‐organized into multiple components, such as hair follicles. Despite their impressive complexity, PSOs are currently generated in the absence of 3D extracellular matrix (ECM) signals and have several major limitations, including an inverted anatomy (e.g., epidermis inside/dermis outside). In this work, a method is established to generate PSOs effectively in a chemically‐defined 3D ECM environment. After examining various dermal ECM molecules, it is found that PSOs generated in collagen ‐type I (COLI) supplemented with laminin 511 (LAM511) exhibit increased growth compared to conventional free‐floating conditions, but fail to induce complete skin differentiation due in part to necrosis. This problem is addressed by generating the PSOs in a 3D bioprinted spindle‐shaped hydrogel device, which constrains organoid growth longitudinally. This culture system significantly reduces organoid necrosis and leads to a twofold increase in keratinocyte differentiation and an eightfold increase in hair follicle formation. Finally, the system is adapted as a microfluidic device to create asymmetrical gradients of differentiation factors and improves the spatial organization of dermal and epidermal cells. This study highlights the pivotal role of ECM and morphogen gradients in promoting and spatially‐controlling skin differentiation in the PSO framework.