A magnetic three-dimensional levitated primary cell culture system for the development of secretory salivary gland-like organoids.

Salivary gland (SG) hypofunction and oral dryness can be induced by radiotherapy for head and neck cancers or auto-immune disorders. These are common clinical conditions that involve loss of saliva-secreting epithelial cells. Several oral complications arise with SG hypofunction that interfere with routine daily activities like chewing, swallowing and speaking. Hence, there is a need for replacing these saliva-secreting cells. Recently, researchers have proposed to repair SG hypofunction via various cell-based approaches in three-dimensional (3D) scaffold-based systems. However, majority of the scaffolds used cannot be translated clinically due to the presence of nonhuman-based substrates. Herein, saliva-secreting organoids/mini-glands were developed using a new scaffold/substrate-free culture system named magnetic 3D levitation (M3DL), which assembles and levitates magnetized primary SG-derived cells (SGDC), allowing them to produce their own extracellular matrices. Primary SGDC were assembled in M3DL to generate SG-like organoids in well-established SG epithelial differentiation conditions for 7 days. After such culture time, these organoids consistently presented uniform spheres with greater cell viability and pro-mitotic cells, when compared to conventional salisphere cultures. Additionally, organoids formed by M3DL expressed SG-specific markers from different cellular compartments: acinar epithelial including adherens junctions (NKCC1, Cholinergic muscarinic receptor type 3, E-cadherin, EpCAM); ductal epithelial and myoepithelial (Cytokeratin 14, α-Smooth muscle actin), and neuronal (β3-tubulin, Vesicular acetylcholine transferase). Lastly, intracellular calcium and α-amylase activity assays showed functional organoids with SG-specific secretory activity upon cholinergic stimulation. Thus, the functional organoid produced herein indicate that this M3DL system can be a promising tool to generate SG-like mini-glands for SG secretory repair.