Differentiation of Primary Human Submandibular Gland Cells Cultured on Basement Membrane Extract

Abstract

There is no effective treatment for the loss of functional salivary tissue after irradiation for head and neck cancer or the autoimmune disease Sjögren's syndrome. One possible approach is the regeneration of salivary glands from stem cells. The present study aimed to investigate whether small pieces of human submandiblar gland tissue contain elements necessary for the reconstruction of salivary rudiments in vitro via acinar and ductal cell differentiation. Primary submandibular gland (primary total human salivary gland; PTHSG) cells were isolated from human tissue and cultured in vitro using a new method in which single cells form an expanding epithelial monolayer on plastic substrates. Differentiation, morphology, number, and organization of these cells were then followed on basement membrane extract (BME) using RNA quantitation (amylase, claudin-1 (CLN1), CLN3, kallikrein, vimentin), immunohistochemistry (amylase and occludin), viability assay, and videomicroscopy. On the surface of BME, PTHSG cells formed acinotubular structures within 24 h, did not proliferate, and stained for amylase. In cultures derived from half of the donors, the acinar markers amylase and CLN3 were upregulated. The PTHSG culture model suggests that human salivary gland may be capable of regeneration via reorganization and differentiation and that basement membrane components play a crucial role in the morphological and functional differentiation of salivary cells.

Implantable Three-Dimensional Salivary Spheroid Assemblies Demonstrate Fluid and Protein Secretory Responses to Neurotransmitters

“…Additionally, most of these 3D salivary cultures used MatrigelÔ, collagen I, or other animal-derived products for their scaffolds. 9,[38][39][40][41] The human-compatible 3D culture system reported here supports the growth of organized spherical structures that merge and proliferate to form larger acini-like structures with a central lumen and can be maintained long-term in vitro. These stable spheroid structures expressed essential salivary biomarkers and neurotransmitter receptors.…”

“…Groups that reported salivary acini-like structures or spheroids growing in 2D or 2.5D [34][35][36][37][38] obtained less wellorganized salivary structures compared to those seen in 3D cultures.…”

Perlecan Domain IV Peptide Stimulates Salivary Gland Cell AssemblyIn Vitro

“…3,4 Recent studies have reported the successful isolation of human salivary acinar cells that express many essential markers. 5,6 Joraku et al reported reconstitution of salivary units that expressed a-amylase and an array of tight junction markers in a culture system consisting of collagen and MatrigelÔ. 5 Although promising, this system cannot be used for human tissue engineering because Matrigel, being mouse derived, is not compatible with human systems.…”

“…Recently, it was reported that a similar isolation of acinar cells required the use of animal serum, which promotes the growth of fibroblasts that often overtake epithelial cell cultures. 6 Although their cultures formed acinotubular-type structures when grown on basement membrane extract (BME), the murine-derived BME cannot be used for tissue engineering in humans. 6 Our study delineates a human-compatible system for the differentiation of human salivary gland cells into functional salivary units.…”

“…6 Although their cultures formed acinotubular-type structures when grown on basement membrane extract (BME), the murine-derived BME cannot be used for tissue engineering in humans. 6 Our study delineates a human-compatible system for the differentiation of human salivary gland cells into functional salivary units. To differentiate, cells require cues from several factors, including their extracellular matrix (ECM), growth factors, and integrin-mediated cell-cell interactions.…”