Establishment of functional epithelial organoids from human lacrimal glands

Abstract Background: Tear deficiency due to dysfunction of the lacrimal gland (LG) is one of the major causes of dry eye disease (DED). Therefore, LG stem cell-based therapies have been extensively reported to regenerate injured lacrimal tissue, but the number of stem cells in the LG tissue is pretty low, and 2D long-term cultivation results in the reduction of the differentiation capacity of stem cells. Whereas, 3D LG organoids could be an alternative for a DED therapeutic method, because it is capable of prolonged growth while maintaining the characteristics of the LG tissue. In this study, we developed LG organoids and applied them as cell therapeutics. Methods: Digested cells from human LG tissue were mixed with Matrigel and cultured in five different media modified from human prostate/salivary organoid culture media. After organoid formation, the growth, expression of specific markers and histological characters were analyzed to prove the LG organoids. And then, the secretory function of LG organoids was confirmed by calcium influx or proteomics after pilocarpine treatment. To explore the curability of developed organoids, finally, mouse-derived lacrimal gland organoids were fabricated and transplanted into mouse lacrimal tissue with DED. Results: The histological features and specific marker expression of LG organoids were similar to normal LG tissue. In the pilocarpine treated LG organoid, internal Ca2+ ions and β -hexosaminidase as known a lysosomal protein in tear fluid was increased. Also, the secreted proteins from pilocarpine treated lacrimal organoid were identified through proteomics. More than 70% of the identified proteins were proven to exosome through GO analysis. These results indicate that our developed organoid has a reactivity to pilocarpine, which shows the function of the lacrimal gland. Additionally, we developed LG organoids from Sjogren’s syndrome patients (SS) and confirmed that their histological feutures similar to that of SS-derived LG tissue. Finally, we confirmed that organoids were well engrafted in the lacrimal tissue at 2 weeks after transplantation of mouse LG orgnoid. Conclusion: This current study demonstrate that our established lacrimal gland organoids resemble characteristics of normal lacrimal gland tissue and could be used as cell therapy for patients with dry eye syndrome.