Organotypic Culture of Mouse Meibomian Gland: A Novel Model to Study Meibomian Gland Dysfunction In Vitro

Abstract

PURPOSE. Meibomian glands are essential in maintaining the integrity and health of the ocular surface. Meibomian gland dysfunction (MGD), mainly induced by ductal occlusion, is considered as the major cause of dry eye disease. In this study, a novel in vitro model was established for investigating the role of inflammation in the process of MGD. METHODS. Mouse tarsal plates were removed from eyelids after dissection and explants were cultured during various time ranging from 24 to 120 hours. Meibomian gland epithelial cells were further enzymatically digested and dissociated from tarsal plates before culturing. Both explants and cells were incubated in different media with or without serum or azithromycin (AZM). Furthermore, explants were treated with IL-1β or vehicle for 48 hours. Analyses for tissue viability, histology, biomarker expression, and lipid accumulation were performed with hematoxylin and eosin (H&E) staining, immunofluorescence staining, and Western blot. RESULTS. Higher viability was preserved when explants were cultured on Matrigel with immediate addition of culture medium. The viability, morphology, biomarker expression, and function of meibomian glands were preserved in explants cultured for up to 72 hours. Lipid accumulation and peroxisome proliferator-activated receptor γ (PPARγ) expression increased in both explants and cells cultured in media containing serum or AZM. Treatment with IL-1β induced overexpression of Keratin (Krt) 1 in meibomian gland ducts. CONCLUSIONS. Intervention with pro-inflammatory cytokine IL-1β induces hyperkeratinization in meibomian gland ducts in vitro. This novel organotypic culture model can be used for investigating the mechanism of MGD.

Hedgehog Signaling Pathway Regulates the Proliferation and Differentiation of Rat Meibomian Gland Epithelial Cells

“… 18 , 19 Lipid secretion is an important character of the maturation of meibocytes. 28 Therefore, we confirmed the cells we cultured were meibomian gland epithelial cells, and proliferation and differentiation models of rat meibomian gland epithelial cells in vitro were established.…”

“…The most common pathogenesis of MGD is ductal epithelial hyperkeratinization 28 , 30 and acinar atrophy. 31 Here, we focused on the role of the Hedgehog signaling pathway in the proliferation and differentiation of acini attempting to provide a new target for MGD treatments.…”

“…Previous researchers have established several culture systems for meibomian gland epithelial cells derived from the rabbit, rat, mouse, and human, however, they often take the method of digesting the entire meibomian gland, 13 , 15 , 16 , 27 , 28 which makes the acinar cells mixed with ductal cells. In our study, rat acinar cells were manually separated from ducts, and ducts were discarded rendering the acinar cells we cultured purer.…”

Hyperkeratinization and Proinflammatory Cytokine Expression in Meibomian Glands Induced byStaphylococcus aureus

“…The culture of MG explants was based on a previous protocol 16 with some minor changes. Briefly, MG explants were placed on 24-well plates with the conjunctival side up and cultured in Defined keratinocyte serum-free medium (DKSFM; 10744019; Thermo Fisher Scientific) without using any antibiotics.…”

Three-Dimensional Human Cell Culture Models to Study the Pathophysiology of the Anterior Eye

“…However, ducts are also important in the correct functioning of these glands. No human model developed to date has included meibomian gland ducts, although both acini and ducts were studied using an organotypic culture of mouse meibomian glands [ 48 ]. In this model, the authors cut tarsal plates into segments, plated them on Matrigel ® , and kept them alive and functional for up to 72 h. With this model, the authors were able to show that after the administration of proinflammatory cytokine IL1β, acini cells accumulated lipids, and the ducts underwent keratinization.…”