Encapsulation of primary salivary gland cells in enzymatically degradable poly(ethylene glycol) hydrogels promotes acinar cell characteristics

Abstract

Radiation therapy for head and neck cancers leads to permanent xerostomia due to the loss of secretory acinar cells in the salivary glands. Regenerative treatments utilizing primary submandibular gland (SMG) cells show modest improvements in salivary secretory function, but there is limited evidence of salivary gland regeneration. We have recently shown that poly(ethylene glycol) (PEG) hydrogels can support the survival and proliferation of SMG cells as multicellular spheres in vitro. To further develop this approach for cell-based salivary gland regeneration, we have investigated how different modes of PEG hydrogel degradation affect the proliferation, cell-specific gene expression, and epithelial morphology within encapsulated salivary gland spheres. Comparison of non-degradable, hydrolytically-degradable, matrix metalloproteinase (MMP)-degradable, and mixed mode-degradable hydrogels showed that hydrogel degradation by any mechanism is required for significant proliferation of encapsulated cells. The expression of acinar phenotypic markers Aqp5 and Nkcc1 was increased in hydrogels that are MMP-degradable compared with other hydrogel compositions. However, expression of secretory acinar proteins Mist1 and Pip was not maintained to the same extent as phenotypic markers, suggesting changes in cell function upon encapsulation. Nevertheless, MMP- and mixed mode-degradability promoted organization of polarized cell types forming tight junctions and expression of the basement membrane proteins laminin and collagen IV within encapsulated SMG spheres. This work demonstrates that cellularly remodeled hydrogels can promote proliferation and gland-like organization by encapsulated salivary gland cells as well as maintenance of acinar cell characteristics required for regenerative approaches. Investigation is required to identify approaches to further enhance acinar secretory properties.

Bottom-up assembly of salivary gland microtissues for assessing myoepithelial cell function

“…Alternatively, spheroids can be produced via suspension culture using non-adhesive tissue culture plates. [38] This method suffers from poor control over cell density, spheroid size/shape and cellular organization. It does not support the establishment of in vitro models with a well-defined spatial arrangement of multiple cell types.…”

Salivary gland cell aggregates are derived from self-organization of acinar lineage cells

“…Following one wash with PBS, tissue was incubated for 5 minutes with 0.05% w/v Trypsin-EDTA and strained through a 40 μm filter. Serum-free cell culture media was made as described previously (Shubin et al, 2015; Shubin et al, 2017). Briefly, basal media consisted of 1:1 Dulbecco’s Modified Eagle Medium (DMEM) and F12 (GIBCO), supplemented with Glutamax (1X ; GIBCO), antibiotic solution (100 IU/mL penicillin, 100mg/mL streptomycin, 0.25 mg/mL amphotericin B; GIBCO), N2 supplement (1X Invitrogen), 10 mg/mL insulin (Life Technologies), 1 mM dexamethasone (Sigma), 20 ng/mL epidermal growth factor (EGF; Life Technologies), and 20 ng/mL basic fibroblast growth factor (bFGF; Life Technologies).…”

“…The expansion of a single salivary gland cell following Wnt stimulation has been shown to give rise to multicellular spheres or ‘salispheres’ (Maimets et al, 2016). However, the term ‘salisphere’ has been applied more liberally to all cell aggregates that form within 48 hours following culture of dissociated gland cells (Lombaert, Brunsting, Wierenga, Faber, et al, 2008; Maimets et al, 2016; Pringle, Van Os, & Coppes, 2013; Shubin et al, 2015; Shubin et al, 2017). In fact, the use of sphere assays as an indicator of stem cells in neuron cultures has been challenged, with timelapse data providing direct demonstration that self-organization contributes to the increase in size of cultured neural spheres (Mori et al, 2006; Singec et al, 2006).…”

Comparing human and mouse salivary glands: A practice guide for salivary researchers

“…Regarding polymers and hydrogels, chemically defined and biologically inert compounds have shown great potential for supporting primary SG cell growth while minimizing the concerns for tumorigenesis upon implantation. For example, enzymatically degradable poly (ethylene glycol) hydrogels support an acinar cell phenotype in primary mouse SMG cells, as demonstrated by maintained expression of the markers AQP5 and Nkcc1 (Shubin et al, 2017). Additionally, hydrogels offer more flexibility by providing the option to plate cells on top of or to encapsulate them within a gel (Shubin et al, 2017, Pradhan-Bhatt et al, 2013).…”