Lacrimal gland spheroids in tissue specific hydrogel for dry eye modelling

A profound understanding of the physiology of the lacrimal gland is essential for developing regenerative therapies for aqueous deficient dry eye syndrome (ADDE). This study presents a reproducible, high-throughput method to generate functional spheroids of the lacrimal gland as building blocks for a three-dimensional (3D) in vitro model. Co-culturing primary lacrimal gland epithelial cells (EpC), mesenchymal stromal cells (MSC), and human umbilical vein endothelial cells (HUVEC) in non-adhesive agarose microwells resulted in rapid spheroid formation within 24 h. These spheroids developed a defined spatial organization, with EpC forming an outer sheath, HUVEC adopting a tube-like structure, and MSC interspersed between them. Compared to 2D-cultures, lacrimal gland spheroids exhibited lower viability but enhanced secretory function, as indicated by β-hexosaminidase activity. Embedding of lacrimal gland spheroids in decellularized lacrimal gland hydrogel (dLG-HG) or tissue-unspecific collagen-I improved both viability and secretory activity, with dLG-HG showing superior effects. Embedding led to a reorganization of the cell types so that EpC formed a central lumen resembling an acinar structure. This study demonstrates that combining 3D spheroids with a tissue-specific ECM scaffold creates a robust, physiologically relevant lacrimal gland in vitro model. Such a model provides a valuable platform for elucidating the pathomechanisms of lacrimal gland dysfunction and for screening potential regenerative therapies to restore tear secretion in patients with ADDE.