Fostering cell–cell interactions and integrating angiocrine factors to promote the development of salivary microtissues in <scp>3D</scp>

Abstract Salivary gland development requires vascular input signals. However, whether endothelial cell‐secreted angiocrine factors have any inductive effects on adult human salivary gland stem/progenitor cells (hS/PCs) is unknown. To advance the engineering of functional salivary glands, we probed the effects of epithelial and endothelial crosstalk on the growth and differentiation of hS/PCs. Culture of hS/PCs in agarose microwells led to the formation of multicellular spheroids with close cell–cell contacts. Compared with the day 3 culture maintained in the hepatocyte media (HEP) typically used for hS/PCs, 7‐day culture in the endothelial cell growth media (EMG2), significantly increased the expression of genes encoding the E‐cadherin ( CDH1 ), stem/progenitor markers ( KRT5 , KRT14 , MYC ), acinar markers ( AQP5 , AMY, SLC12A1 ), and extracellular matrix proteins ( LAMA1 , FN1 ). Subsequent cultivation of hS/PC spheroids in a hyaluronic acid (HA)‐derived, cell‐adhesive, and proteolytically degradable hydrogel yielded hydrogel‐encapsulated microtissues with complex, multilobulated structures, consisting of fibronectin‐encased lobules connected by F‐actin structures. Addition of a CD31+/vWF+ endothelial cell monolayer on top of the hS/PC‐laden gel construct led to the development of salivary gland microtissues containing differentiated cells expressing key acinar and ductal cell markers. In vivo work showed that the cell‐free HA gels implanted in the partially resected rat parotid gland were degraded in 21 days and did not adversely affect the native tissue structure. Collectively, fostering epithelial cell–cell interaction and integrating endothelial cell‐secreted angiocrine signals led to the development of pro‐acinar salivary gland mimetics from adult salivary gland stem/progenitor cells.