(J-386) Bioengineering Salivary Gland Spheroids and Organoids for High Content Screening

Introduction:: Salivary glands (SGs) are exocrine organs responsible for the production and secretion of saliva. Xerostomia or dry mouth is a SG disorder affecting millions of people worldwide and can cause mild oral discomfort to severe dry mouth affecting the overall health and quality of life. Among several reasons, Sjogren’s syndrome and radiotherapy-induced xerostomia in patients with head and neck cancers are the top causes of dry mouth. Currently, there is no permanent therapeutic solution for the treatment of xerostomia. The only FDA-approved drug, amifostine, causes significant side effects in patients (1). Current drug development pipelines are complex, lengthy, and expensive and thus there is a need to fast-track the process to identify therapeutics to treat xerostomia. SG in vitro and ex vivo models have played a critical role in our understanding of the SG pathophysiology (2). Nevertheless, recapitulating the complex architecture of SG possess inherent challenges like the lack of stable human cell lines, slow cell proliferation rate, and the inability of primary single cells to reorganize well in three-dimensional (3D) culture. Additionally, there are challenges in developing mechanically and biochemically tunable matrices to support functionally and phenotypically heterogeneous cells in the SGs. Herein, we use alginate-gelatin-hyaluronic acid-based hydrogels to generate physiologically and functionally relevant SG spheroids and organoids. These 3D models can be combined with multi-well, microfluidic platforms to potentially test hundreds of drugs and biomolecules to screen therapeutic candidates that can restore lost SG function.


Materials and Methods:: To prepare alginate (A), gelatin (G), and hyaluronic acid (HA) hydrogels, briefly, sodium alginate and bovine gelatin powders were dispersed into sterile PBS and stirred for 3 h at 50°C/500 rpm. Then, HA was incorporated into AG composites at 30 °C and mixed for 30 mins to prepare the final AGHA hydrogels. For spheroid cultures, SG cell lines were seeded at a concentration of 2 x 10⁶ cells per ml of hydrogel, and hydrogel 3D models were generated using the hanging drop technique and cultured for 14-50 days. Live/dead assay, immunofluorescence staining, and reverse transcriptase – quantitative polymerization chain reaction (RT-qPCR) were performed to assess spheroid viability, protein, and gene expression, respectively. To establish SG spheroids as irradiation models, we irradiated them with a 15 Gy single dose and tested the effect of radioprotective agent WR-1065. Cell metabolic assay (WST-8) and immunostaining of γH2AX, caspase-3, and Ki-67 markers were performed to analyze the irradiated spheroids. For optimizing organoid culture, human or mouse SG were enzymatically digested to obtain SG cell clusters. The clusters were grown in suspension culture for 2-3 days under defined culture medium supplemented with growth factors. The cell clusters were then collected and mixed with AGHA hydrogels and seeded as described before. Live/dead assay, immunostaining, RT-qPCR, and histological evaluation of organoids were performed to confirm their ability to capture features of native tissue.

Results, Conclusions, and Discussions:: We performed a live-dead assay of our AGHA-cultured spheroids and organoids to assess their ability to proliferate and maintain long-term viability. We successfully cultured human SG spheroids with high viability for ~ 50 days and mice organoids for ~75 days (Fig.1A). Our spheroid cultures showed distinct glandular (acinar) and tubular-like (ductal) structures in 3D culture depending on the cell type in addition to the lumen forming organoids in long-term cultures. Immunofluorescence staining revealed a robust localization of key SG markers like AQP5, NKCC-1, and ZO-1, progenitor markers like CK5, and cell-ECM regulators like β-integrin and vinculin (Fig.1B). Further, PAS and H&E staining confirmed the presence of mucin positive cells in lumen forming organoids (Fig.1C). Irradiation experiments confirmed double-stranded (ds) DNA breaks and apoptosis in SG spheroids by expressing high levels of γH2AX and caspase 3 markers respectively. Further, treatment with radioprotective agent WR-1065, an active form of amifostine, limits the dsDNA breaks and improves overall cell metabolic activity in SG spheroids. In this work, we showed that AGHA hydrogels serve as an ideal platform for the generation and expansion of multicellular SG spheroids. We also found that SG spheroids, depending on the SG cell line, re-organized into acinar- and or ductal-like structures in 3D culture. Further, when complemented with growth factors and soluble cues, primary SG cell clusters reorganize into lumen-forming organoids and show robust expression of makers identifying the different cell populations in native SG tissue. We also show that AGHA hydrogels are versatile bio inks for 3D culture applications owing to their simple mechanism of ionic crosslinking using cations like Ca+ and their reversal by use of chelators like citrates to isolate viable spheroids and organoids with minimal loss for downstream analysis.

Our organoids and spheroids have shown evidence as promising SG microtissues that can be combined with multiwell plates for use in high throughput and high content screening of radioprotective agents and therapeutic biomolecules. The physiological relevance of these spheroids and organoids can be further improved by culturing them under dynamic conditions in combination with microfluidic devices and organ-on-chip platforms.


Acknowledgements (Optional): : This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) grant # 03615. Part of presenting author's stipend is supported by Fonds de Recherche du Québec - Santé (FRQS) doctoral award # 304367.

References (Optional): : 1) D. Rades et al., Serious adverse effects of amifostine during radiotherapy in head and neck. cancer patients. Radiotherapy and Oncology 70, 261-264 (2004). 2) J. Iyer et al., Salivary gland bioengineering-yesterday, today, tomorrow! Histology and Histopathology, 18580-18580 (2023).