INTRODUCTION: Development of a tissue-engineered,salivary bio-gland will benefit patients suffering from xerostomia due to loss of fluid-secreting acinar cells.This study was conducted to develop a bioengineering system to generate glandular organoids from human salivary glandular stem cells(hSGSCs)through three-dimensional(3D)spheroid culture.METHODS & RESULTS: Microwells were fabricated by photopatterning of PEG hydrogel in the presence of an electrospun PCL nanofibrous scaffold.hSGSCs were plated on plastic dishes,PCL nanofibers,or PCL nanofibrous microwells.Upon a differentiation induction,hSGSCs cultured on PCL nanofiber and PCL microwells aggregated to form 3D acinar-like organoids and the greatest differentiation potency was observed on the PCL microwells.The 3D-assembled organoids in the PCL microwells expressed higher levels of salivary epithelial markers(α-amylase and AQP5)and tight junction proteins(ZO-1 and occludin)than those on the PCL.Furthermore,the 3D-assembled organoids in the PCL microwells showed higher levels of transepithelial electrical resistance(TER),α-amylase secretion and intracellular calcium concentration([Ca2+]i)than those on the PCL nanofibers,suggesting more robust and functional organization to polarized salivary acinar units from hGSCs.DISCUSSION & CONCLUSIONS: We established a bioengineering 3D spheroid culture system to generate robust and functional acinar-like organoids from hSGSCs.PCL nanofibrous microwells can be applied in the future for bioengineering of an artificial bio-salivary gland for restoration of SG function.