3D biomimetic culture enables meibomian gland cell spheroids to recapitulate in vivo-like lipid synthesis patterns via mechanical cues

Meibomian gland dysfunction (MGD) is a leading cause of evaporative dry eye disease, characterized by insufficient lipid secretion from meibocytes. Although immortalized human meibomian gland epithelial cells (iHMGECs) are widely used, conventional two-dimensional (2D) cultures fail to recapitulate key features of meibocyte differentiation, particularly lipid synthesis, limiting mechanistic studies and therapeutic development. This study aimed to establish a simple and scalable three-dimensional (3D) in vitro model that supports functional lipidogenesis of iHMGECs, while enabling mechanistic investigation and pharmacological evaluation relevant to MGD. iHMGECs were cultured on uniform gelatin methacryloyl (GelMA)-based microcarriers to form MG cell spheroids. Lipid synthesis and differentiation were assessed by gene expression analysis, immunofluorescence, and lipid staining. Transcriptomic profiling was performed to identify pathway-level changes induced by 3D culture. Mechano-transduction signaling was examined through cytoskeletal organization and YAP/TAZ activity, and functional validation was conducted using pharmacological modulation. The responsiveness of the model to the PPARγ agonist rosiglitazone was evaluated to assess its utility for drug screening. MG cell spheroids maintained high viability and exhibited markedly enhanced lipid synthesis compared with 2D cultures, as evidenced by robust upregulation of PPARγ, PLIN2, and AWAT2, along with abundant neutral lipid accumulation. Transcriptomic analysis revealed enrichment of extracellular matrix, lipid metabolic, and differentiation-related pathways, partially overlapping with chemically induced differentiation. Mechanistically, 3D culture attenuated mechano-transduction signaling, characterized by reduced YAP/TAZ activity and altered cytoskeletal tension, favoring lipidogenic differentiation. Importantly, MG cell spheroids displayed significantly increased sensitivity to rosiglitazone, demonstrating amplified lipidogenic responses relative to 2D cultures. This biomaterial-enabled MG cell spheroid platform effectively reproduces key lipid-secreting functions of meibocytes in vitro and provides a physiologically relevant, scalable model for studying MGD mechanisms and screening lipid-modulating therapeutics.