(432e) HA-Based Hydrogels for Controlled Culture of Salivary Gland Organoids

Objective: Salivary gland (SG) dysfunction results in limited or complete loss of saliva production, leading to xerostomia. This is caused by both complications of autoimmunity (Sjogren’s Syndrome) and side effects from cancer radiation therapy. The cells most affected are the acinar cells, which are responsible for producing and secreting saliva. Without saliva, patients not only experience loss of lubrication in the oral cavity but also decreased immune defense and tissue loss due to the reduction of salivary antimicrobial properties, leading to a decreased quality of life and risk for systemic infection. Current treatments are superficial, including artificial saliva, which provides only temporary relief; however, regenerative medicine may enable more efficient and permanent solutions toward regaining SG function. Many groups utilize Matrigel, a tumor-derived matrix, for 3D culture, but this offers no control over culture conditions, and is not clinically translatable. To this end, we have developed a novel differentiation strategy based on salivary gland (SG) development to obtain SG epithelial progenitors from induced pluripotent cells with no need for virus-mediated gene overexpression or embryoid body microsurgery. In this study, we aimed to design a hyaluronic acid (HA)-based hydrogel to generate polarized SG organoids in a matrix with controlled mechanical properties and biochemical cues.

Methods: HA was conjugated to bicycle[6.1.0]non-4-yn-9-ylmethanol (BCN) and assessed with NMR to ensure chemical purity. The crosslinker components, PEG azide aldehyde (CHO) and PEG azide hydroxylamine (ONH2) were bound in a carbonyl reaction forming the crosslinker, PEG diazide. When the modified HA and PEG diazide are mixed, the components crosslink via a biorthogonal strain-promoted alkyne-azide cycloaddition (CLICK) reaction, forming the hydrogel. A range of crosslinker densities was tested with oscillatory rheology to measure the mechanical properties of the hydrogel and determine the optimal stiffness for organoid growth. Peptides RGD, YIGSR, IKVAV, and TWSKV were modified with an amine group at the N-terminus to conjugate with the modified HA to enable cell attachment and organoids formation. iPSCs were cultured until 70% confluent before differentiation commenced. The cells were subjected to a series of growth factors, serum, and inhibitor for 17 days at which point a monolayer was formed. At day 17 (D17) cells were trypsinized with TryplE and the cells were placed in the hydrogel and coaxed to differentiate in a series of steps mimicking SG development.

Results and Conclusion: The synthesized hydrogel was subjected to frequency and amplitude sweeps, to measure the storage and loss moduli. Furthermore, the shear-thinning and self-healing abilities of the hydrogel were tested with an alternating time sweep test in which we observed the change in viscosity of the HA gel during period of high and low stress. Repeatedly, the hydrogel showed reduced viscosity at high stress and return to its original viscosity at low stress, demonstrating shear-thinning and self-healing behavior.

After 17 days of differentiation, the identity of SGEP progenitors was verified with RT-PCR, immunostaining and flow cytometry for key transcription factors, such as FoxC1 and Sox9. The stiffness of the HA-based hydrogel depended significantly on the crosslinker density, with lower densities, such as 2.5wt%, allowing greater organoid growth. Although, the addition of peptides did not change the mechanical properties of the hydrogel, it affected the biological environment, as certain combinations allowed clear lumen formation and development of function after 30 days in 3D culture. Lumen formation and cell polarization within the organoids was evaluated by immunostaining and confocal imaging for ZO-1 and actin. SGO differentiation was evaluated by immunostaining for key acinar (Mist1, Sox10) and ductal (K8/18, K7) markers; while development of salivary function was assessed by measurements of the calcium flux in response to the acetylcholine analogue, carbachol.

In conclusion, our work showed that a HA-based shear thinning hydrogel with appropriate mechanical properties and biochemical signals support the development of lumenized and functional SGO from iPSC derived SG progenitors. This hydrogel provides a well-controlled and clinically translatable platform for understanding SGO development as well as for drug screening for treatment of hyposalivation disorders due to autoimmunity or radiation therapy.