Induction of meibocyte differentiation by three-dimensional, matrigel culture of immortalized human meibomian gland epithelial cells to form acinar organoids

Purpose: Recent studies have shown that two-dimensional (2D) culture of primary rabbit and immortalized human meibomian gland epithelial cells (iHMGEC) do not recapitulate normal meibocyte differentiation and fail to express critical enzymes necessary for synthesis of meibum lipids. The purpose of this study was to test the hypothesis that 3D-spheroid culture of iHMGEC can facilitate meibocyte differentiation and induce the expres-sion of acyl-CoA wax-alcohol acyltransferase 2 (AWAT2), shown to be required for synthesis of meibum wax esters.Methods: iHMGEC were suspended in matrigel/basement membrane matrix and grown in proliferation media to form distinct cell clusters or spheroids. Cells were then treated with serum-free, differentiation media (advanced DMEM/F12) with and without FGF10 and synthetic agonists for the nuclear lipid receptor, peroxisome pro-liferator activator receptor gamma (PPAR gamma). Cells were then evaluated for differentiation markers using western blotting, immunocytochemistry (ICC) and real-time PCR. Control cells were grown in standard 2D culture systems.Results: Under proliferative conditions, 3D culture induced the formation of symbolscript spheroids that contained a symbolscript undifferentiated, basal cell population. When spheroids were switched to differentiation media containing PPAR gamma agonists, two different organoid populations were detected, a KRT6low population that was symbolscript and a KRT6high population that was AWAT2-/PPAR gamma-, suggesting that iHMGEC exhibit a dual differentiation potential toward either a ductal or meibocyte organoid phenotype.Conclusion: The 3D culturing of iHMGEC can induce the formation of both meibocyte and ductal organoids and may thus serve as a better in vitro model system for studying the regulatory mechanisms controlling meibomian gland function.