Differentiation Patterns of Immortalized Human Meibomian Gland Epithelial Cells in Three-Dimensional Culture.

Abstract

To establish a simplified three-dimensional (3D) meibomian gland culture model using a meibomian gland epithelial cell (HMGEC) line that might be a useful tool to gain deeper insights into meibomian gland dysfunction. For this purpose, 3D differentiation patterns and growth characteristics of HMGECs were studied on various membranes/scaffolds as well as in hanging drops. Several types of inserts consisting of different materials (Millicell-HA, Millicell-PCF, ThinCert, and Alvetex) as well as hanging drop culture were analyzed. Culture conditions were optimized employing exposure to air (air-lift) and different cell culture media for a maximum of 28 days. To characterize cell differentiation in the developed 3D model, the expression pattern of cytokeratins was investigated by immunohistochemistry. Sudan III staining was performed for detection of lipid formation and transmission electron microscopy (TEM) was used for ultrastructural analysis. Only Alvetex scaffolds and the hanging drop method revealed satisfactory results with regard to 3D culture. Continuous use of proliferation medium (serum-free keratinocyte medium containing epidermal growth factor and bovine pituitary extract) and air-lift were important steps for HMGEC differentiation in 3D culture. However, HMGECs only reached a differentiating state and never became mature or hypermature. When cultured in hanging drops, HMGECs showed serum-induced keratinization processes. HMGECs have the capability to differentiate in a long-term 3D culture, especially when adapted to an air-rich environment. However, even in the 3D format, HMGECs only reach a state of differentiating meibocytes.