Cell culture of differentiated human salivary epithelial cells in a serum‐free and scalable suspension system: The salivary functional units model

Abstract

Saliva aids in digestion, lubrication, and protection of the oral cavity against dental caries and oropharyngeal infections. Reduced salivary secretion, below an adequate level to sustain normal oral functions, is unfortunately experienced by head and neck cancer patients treated with radiotherapy and by patients with Sjögren's syndrome. No disease‐modifying therapies exist to date to address salivary gland hypofunction (xerostomia, dry mouth) because pharmacotherapies are limited by the need for residual secretory acinar cells, which are lost at the time of diagnosis, whereas novel platforms such as cell therapies are yet immature for clinical applications. Autologous salivary gland primary cells have clinical utility as personalized cell therapies, if they could be cultured to a therapeutically useful mass while maintaining their in vivo phenotype. Here, we devised a serum‐free scalable suspension culture system that grows partially digested human salivary tissue filtrates composing of acinar and ductal cells attached to their native extracellular matrix components while retaining their 3D in vivo spatial organization; we have coined these salivary spheroids as salivary functional units (SFU). The proposed SFU culture system was sub‐optimal, but we have found that the cells could still survive and grow into larger salivary spheroids through cell proliferation and aggregation for 5 to 10 days within the oxygen diffusion rates in vitro. In summary, by using a less disruptive cell isolation procedure as the starting point for primary cell culture of human salivary epithelial cells, we demonstrated that aggregates of cells remained proliferative and continued to express acinar and ductal cell‐specific markers.

Insight into Salivary Gland Aquaporins

“…However, when these human salivary cells were enzymatically digested and plated on three-dimensional (3D) gels or in polarized cell monolayers, AQP5 expression was either lost or its localization was shifted to the cytoplasm [ 188 , 189 , 190 ]. Recently, we have devised a new strategy by using a serum-free scalable suspension culture system that grows partially digested human salivary tissue filtrates composed of acinar and ductal cells attached to their native extracellular matrix components, while retaining their 3D in vivo spatial organization [ 191 ]. We demonstrated that aggregates of cells remained proliferative and continued to express acinar (such as AQP5) and ductal cell-specific markers for 5 to 10 days [ 191 ].…”

“…Recently, we have devised a new strategy by using a serum-free scalable suspension culture system that grows partially digested human salivary tissue filtrates composed of acinar and ductal cells attached to their native extracellular matrix components, while retaining their 3D in vivo spatial organization [ 191 ]. We demonstrated that aggregates of cells remained proliferative and continued to express acinar (such as AQP5) and ductal cell-specific markers for 5 to 10 days [ 191 ]. We have also reported that two cell surface markers, CD44 and CD166, used in the isolation of human mesenchymal stem cells could be co-localized with AQP5-positive serous (CD44) and mucous (CD166) acinar cells [ 192 ].…”

Encapsulation of Primary Salivary Gland Acinar Cell Clusters and Intercalated Ducts (AIDUCs) within Matrix Metalloproteinase (MMP)‐Degradable Hydrogels to Maintain Tissue Structure and Function

“…Primary AIDUCs cells were isolated, as previously described, with minor modifications. [35,36] Briefly, female C57BL/6 mice aged 8-12 weeks were euthanized according to animal protocols approved by the University of Rochester University Committee for Animal Resources. SMGs were surgically removed, finely minced with a razor blade, and dissociated by incubation in 10 mL Hank's Balanced Salt Solution (HBSS) (Gibco) supplemented with 0.5% BSA, 1 m CaCl 2 , 1 m MgCl 2 , 100 U mL −1 collagenase Type II (Gibco), and 1 mg mL −1 hyaluronidase (Sigma) at 37°C for 60 min with gentle shaking.…”

“…Although the aggregates promoted viability, proliferation, and even exhibited evidence of cellular organization including apicobasolateral organization of acinar markers, there was a loss of acinar cell phenotype during aggregate formation with evidence for acinar transition to ductal phenotypes. [22,30] To maintain differentiated acini, a milder dissociation protocol was adapted to isolate AIDUCs, [35,36] and the size distribution of the freshly isolated AIDUCs, analyzed using light microscopy, was 20-100 μm (Figure S1, Supporting Information). While comprised of both acinar and duct cells (Figure S2A, Supporting Information), most cells within AIDUCs are acinar cells, as indicated by the positive staining of acinar cell markers NKCC1 and AQP5 (Figure S2A-C, Supporting Information).…”

Salivary Gland Tissue Engineering Approaches: State of the Art and Future Directions

“…These ECM proteins and their integrins orch trate events during salivary gland development and homeostasis including branchi morphogenesis, cleft formation, apico-basal polarization, adhesion, growth, and mig Primary cultures from adult tissue typically start with dissociation of the entire gland, containing acinar, duct, and myoepithelial cells. While some groups dissociate glands into single cells [23,26], others have highlighted the importance of maintenance of partial tissue structure to retain cell-cell contacts to promote 3D morphology and polarization [27][28][29][30]. For embryonic cultures, tissues are commonly grown as explants on a membrane with an air-liquid interface [31][32][33], although they can also be dissociated similar to adult tissue.…”