Organotypic 3D Culture in Nanoscaffold Microwells Supports Salivary Gland Stem-Cell-Based Organization

[Image: see text] The self-organizing properties of stem cells have been exploited to generate organoids, organ-specific, cell-containing, three-dimensional (3D) structures. The present study aimed to introduce a novel bioengineering technique for driving the effective organization of adult tissue stem cells via niche-independent 3D microwell culture. Microwells were fabricated by photopatterning poly(ethylene glycol) hydrogel in the presence of an electrospun polycaprolactone nanofibrous scaffold. Human single clonal salivary gland stem cells (SGSCs) were cultured in nanofibrous microwells through two simple steps, priming and differentiation. Before the induction of 3D organization, single clonal SGSCs were preconditioned to aggregate to form 3D spheroids in different matrices, such as Matrigel, floating dish, and microwells. Expression of salivary stem cell markers and pluripotency markers was greater in 3D spheroid cultures than in 2D plastic culture. Lobular structures were organized by changing media, and those in microwells exhibited higher salivary acinar, ductal, and tight junction marker levels and decreased stem-cell marker levels relative to other 3D cultures. Furthermore, higher α-amylase secretion and intracellular calcium levels were observed in the presence of adrenergic or cholinergic agonists, respectively, along with an increased intracellular pH, suggesting more functional salivary organoid formation. These microwell-driven organoids also engrafted successfully into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Our results showed that microwell-cultured SGSCs organize into salivary structures and that this biomimetic 3D culture technique can promote effective generation of niche-independent single stem-cell-based 3D organoids.