BACKGROUND Aqueous-deficient dry eye disease (ADDED) resulting from dysfunction of the lacrimal gland (LG) is currently incurable. Although LG stem/progenitor cell-based therapy is considered to be a promising strategy for ADDED patients, the lack of a reliable serum-free culture method to obtain enough lacrimal gland stem cells (LGSCs) and the basic standard of LGSC transplantation are obstacles for further research. METHODS Adult mouse LGSCs were cultured in Matrigel-based 3D culture under serum-free culture condition, which contained EGF, FGF10, Wnt3A, and Y-27632. LGSCs were continuously passaged over 40 times every 7 days, and the morphology and cell numbers were recorded. LGSCs were induced to differentiate to ductal cells by reducing Matrigel rigidity, while fetal bovine serum was used for the induction of acinar cells. RT-PCR or qRT-PCR analysis, RNA-sequence analysis, H&E staining, and immunofluorescence were used for characterization and examining the differentiation of LGSCs. LGSCs were allotransplanted into diseased LGs to examine the ability of repairing the damage. The condition of eye orbits was recorded using a camera, the tear production was measured using phenol red-impregnated cotton threads, and the engraftments of LGSCs were examined by immunohistochemistry. RESULTS We established an efficient 3D serum-free culture for adult mouse LGSCs, in which LGSCs could be continuously passaged for long-term expansion. LGSCs cultured from both the healthy and ADDED mouse LGs expressed stem/progenitor cell markers Krt14, Krt5, P63, and nestin, had the potential to differentiate into acinar or ductal-like cells in vitro and could engraft into diseased LGs and relieve symptoms of ADDED after orthotopic injection of LGSCs. CONCLUSION We successfully established an efficient serum-free culture for adult mouse LGSCs aiming at LG repair for the first time. Our approach provides an excellent theoretical and technical reference for future clinical research for ADDED stem cell therapy.

中文翻译：

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泪腺干细胞长期无血清培养的建立，旨在修复泪腺。

背景技术由泪腺（LG）的功能障碍导致的缺水性干眼病（ADDED）目前是无法治愈的。尽管基于LG干细胞/祖细胞的治疗被认为是ADDED患者的一种有前途的策略，但缺乏可靠的无血清培养方法来获得足够的泪腺干细胞（LGSC）和LGSC移植的基本标准仍然是障碍。进一步的研究。方法成年小鼠LGSCs在无血清培养条件下，以Matrigel为基础的3D培养，其中包含EGF，FGF10，Wnt3A和Y-27632。LGSC每7天连续传代40次以上，并记录其形态学和细胞数。LGSCs通过降低基质胶的刚性而诱导分化为导管细胞，而胎牛血清则用于诱导腺泡细胞。RT-PCR或qRT-PCR分析，RNA序列分析，H＆E染色和免疫荧光用于表征和检查LGSC的分化。LGSC被同种异体移植到患病的LG中，以检查修复损伤的能力。使用照相机记录眼眶的状况，使用酚红浸渍的棉线测量眼泪的产生，并通过免疫组织化学检查LGSC的植入。结果我们为成年小鼠LGSC建立了有效的3D无血清培养，其中LGSC可以连续传代以进行长期扩增。从健康小鼠和添加的小鼠LG中培养的LGSC表达干/祖细胞标记物Krt14，Krt5，P63和Nestin，有可能在体外分化为腺泡或导管样细胞，并且可以原位注射LGSCs植入患病的LGs并缓解ADDED症状。结论我们成功建立了针对成年小鼠LGSC的有效无血清培养物，首次针对LG修复。我们的方法为ADDED干细胞疗法的未来临床研究提供了极好的理论和技术参考。