Slow hydrogel matrix degradation enhances salivary gland mimetic phenotype.

We recently developed a salivary gland tissue mimetic (SGm), comprised of salivary gland cells encapsulated in matrix metalloproteinase (MMP)-degradable poly(ethylene glycol) hydrogels within arrays of ∼320 µm diameter spherical cavities molded in PDMS. The SGm provides a functional and physiologically relevant platform well-suited to high-throughput drug screening for radioprotective compounds. However, the utility of the SGm would benefit from improved retention of acinar cell phenotype and function. We hypothesized that tuning biochemical cues presented within the PEG hydrogel matrix would improve maintenance of acinar cell phenotype and function by mimicking the natural extracellular matrix microenvironment of the intact gland. Hydrogels formed using slower-degrading MMP-sensitive peptide crosslinkers showed >2-fold increase in sphere number formed at 48 h, increased expression of acinar cell markers, and more robust response to calcium stimulation by the secretory agonist, carbachol, with reduced SGm tissue cluster disruption and outgrowth during prolonged culture. The incorporation of adhesive peptides containing RGD or IKVAV improved calcium flux response to secretory agonists at 14 days of culture. Tuning the hydrogel matrix improved cell aggregation, and promoted acinar cell phenotype, and stability of the SGm over 14 days of culture. Furthermore, combining this matrix with optimized media conditions synergistically prolonged the retention of the acinar cell phenotype in SGm. STATEMENT OF SIGNIFICANCE: Salivary gland (SG) dysfunction occurs due to off-target radiation due to head and neck cancer treatments. Progress in understanding gland dysfunction and developing therapeutic strategies for the SG are hampered by the lack of in vitro models, as salivary gland cells rapidly lose critical secretory function within 24 hours in vitro. Herein, we identify properties of poly(ethylene glycol) hydrogel matrices that enhance the secretory phenotype of SG tissue mimetics within the previously-described SG-microbubble tissue chip environment. Combining slow-degrading hydrogels with media conditions optimized for secretory marker expression further enhanced functional secretory response and secretory marker expression.

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

Progress in the development of salivary gland regenerative strategies is limited by poor maintenance of the secretory function of salivary gland cells (SGCs) in vitro. To reduce the precipitous loss of secretory function, a modified approach to isolate intact acinar cell clusters and intercalated ducts (AIDUCs), rather than commonly used single cell suspension, is investigated. This isolation approach yields AIDUCs that maintain many of the cell-cell and cell-matrix interactions of intact glands. Encapsulation of AIDUCs in matrix metalloproteinase (MMP)-degradable PEG hydrogels promotes self-assembly into salivary gland mimetics (SGm) with acinar-like structure. Expression of Mist1, a transcription factor associated with secretory function, is detectable throughout the in vitro culture period up to 14 days. Immunohistochemistry also confirms expression of acinar cell markers (NKCC1, PIP and AQP5), duct cell markers (K7 and K5), and myoepithelial cell markers (SMA). Robust carbachol and ATP-stimulated calcium flux is observed within the SGm for up to 14 days after encapsulation, indicating that secretory function is maintained. Though some acinar-to-ductal metaplasia is observed within SGm, it is reduced compared to previous reports. In conclusion, cell-cell interactions maintained within AIDUCs together with the hydrogel microenvironment may be a promising platform for salivary gland regenerative strategies.