The lacrimal gland is crucial for maintaining ocular health by producing the aqueous component of the tear film, which hydrates and nourishes the ocular surface. Decreased production of this component results in dry eye disease, a condition affecting over 250 million people worldwide. However, the scarcity of primary human material for studying its underlying mechanisms and the absence of a cell model for human lacrimal gland epithelial cells present significant challenges. Here, we describe the generation of immortalized human lacrimal gland cell lines through the introduction of an SV40 antigen. We successfully isolated and characterized three cell clones from a female lacrimal gland donor, confirming their epithelial identity through genomic and protein analyses, including PCR, RNAseq, immunofluorescence and cultivation in a 3D spheroid model. Our findings represent a significant advancement, providing improved accessibility to investigate the molecular pathogenesis mechanisms of dry eye disease and potential therapeutic interventions. We identified the expression of typical epithelial cell marker genes and demonstrated the cells’ capability to form 2D cell sheets and 3D spheroids. This establishment of immortalized human lacrimal gland cells with epithelial characteristics holds promise for future comprehensive studies, contributing to a deeper understanding of dry eye disease and its cellular mechanisms.

The lacrimal gland produces the tear film’s aqueous component, which moistens and nourishes the ocular surface to maintain eye health. Reduced production of this component leads to dry eye disease, which affects over 250 million people worldwide. Despite the impact on patients, the availability of primary human material to study underlying disease mechanisms is severely limited and there is no cell model available for human lacrimal gland epithelial cells. After insertion of an SV40 antigen into primary human lacrimal gland epithelial cells, we selected, expanded, and characterized three epithelial cell clones from a female lacrimal gland donor. We show their epithelial character at genomic (PCR and RNA _seq ) and protein (immunofluorescence) levels and grow these cells in a 3D cell spheroid model. Here, we report the development of an immortalized human lacrimal gland epithelial cell line that improves accessibility to study the molecular pathogenesis mechanisms of dry eye disease and link them to causal treatments. We show the expression of typical lacrimal gland epithelial cell marker genes (e.g. PAX6, FOXC1, AQP5, CSTB, and CST6) and describe the feasibility of the cells to form 2D cell sheets and 3D cell spheroids. We successfully established immortalized human lacrimal gland cells with epithelial character. In the future, the integration of these cells into larger studies holds great potential for advancing our understanding of dry eye disease and its underlying cellular mechanisms.