Method: Human parotid gland tissue was collected under an IRB approved protocol from surgical patients. Parotid gland cells were isolated from tissue specimens and expanded. A hyaluronic acid (HA)-based hydrogel scaffold and an HA-laminin gel allowed cell culture in 3D. Cells were stained for progenitor cell markers. Self-assembly and structure formation in 3D was assessed by phalloidin and biomarker staining.
Result: Cells in 3D self-assembled to form functional spheroid structures that exceeded 550μm. Cytokeratin 5 (K5), an intermediate filament protein and progenitor cell marker whose progeny are widespread in both ductal and acinar compartments of submandibular glands, was evaluated. Gene and protein expression profiles indicated that K5 was highly expressed among cultured cells in HA hydrogels. Both our expanded progenitor cells and those in 3D expressed stem cell marker c-Kit. Other stem cell markers expressed by these cells included Msi-1 and CD44. Cells seeded in HA-laminin gels differentiated into self-assembled structures that were less organized than spheroids in HA alone. Conditioned media from rat dorsal root ganglia explants accelerated self-assembly and branched structures were observed in laminin gels. K19 staining confirmed the presence of ductal cells among branched structures. Ongoing studies involve replacing laminin with human-compatible bioactive laminin peptides and using perlecan-derived peptide to support vascular ingrowth in modular bilayer hydrogels.
Conclusion: An HA and matrix-modified modular hydrogel culture system that fosters growth and differentiation of human parotid gland progenitor cells into branched structures was established. Ongoing studies involve demonstrating functionality of these structures upon neurotransmitter stimulation and vascular penetration to provide fluid source.