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

Jan 23, 2019Journal of tissue engineering and regenerative medicine

A magnetic 3D system for growing salivary gland-like cell clusters

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Abstract

Saliva-secreting organoids were developed using a new scaffold/substrate-free culture system called magnetic 3D levitation (M3DL).

Magnetic 3D levitation (M3DL) allows primary salivary gland-derived cells to produce their own supporting structures.
After 7 days of culture in M3DL, organoids exhibited uniform spheres with greater cell viability and more pro-mitotic cells compared to traditional salisphere cultures.
The organoids expressed specific markers associated with different types of salivary gland cells, including acinar, ductal, myoepithelial, and neuronal components.
Functional assays demonstrated that these organoids could secrete saliva-related substances in response to stimulation.
The M3DL system may serve as a promising approach for generating salivary gland-like mini-glands for potential repair of salivary gland function.

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Salivary gland (SG) hypofunction and oral dryness can be induced by radiotherapy for head and neck cancers or autoimmune 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 such as 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 non-human-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 (SGDCs), allowing them to produce their own extracellular matrices. Primary SGDCs 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 with 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, and EpCAM); ductal epithelial and myoepithelial (cytokeratin 14 and α-smooth muscle actin); and neuronal (β3-tubulin and 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.

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

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