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

Abstract

PURPOSE. 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.METHODS. 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. RESULTS.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. CONCLUSIONS.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.

Evaluation of Cell Harvesting Techniques to Optimize Lipidomic Analysis from Human Meibomian Gland Epithelial Cells in Culture

“…Importantly, across all of the different harvesting and extraction techniques, the lipidome from HMGECs still shows significant differences relative to the lipidome from normal human meibum. One possibility to explain these discrepant findings is that immortalized HMGECs may fail to fully differentiate in culture, as previously hypothesized by other groups [3,22,23], though the methodology of Schroder et al [23] has recently been challenged [24]. Hampel et al stated that HMGECs that are differentiated by serum reach only an early stage of differentiation [25].…”

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

“…The first 3D culture of meibomian glands was prepared in 2016 using rat cells [45]. In 2018, Asano et al used HMGECs and different biomaterials and techniques to create 3D models of human meibomian glands [46]. First, they used different membranes (Millicell ® -HA, Millicell ® -PCF, and ThinCert™) as substrates on which HMGECs were seeded and cultured in air-lifted conditions.…”

“…In these 3D scaffolds, HMGECs differentiated into sphere-shaped colonies on the apical surface. Finally, the hanging drop technique, initially described by Harrison in 1907 and mentioned at the beginning of this review, was also used to establish 3D cultures of human meibomian glands [46]. Around 1000 cells were seeded in a 20 µL drop of proliferation medium on the inside of petri dish lids.…”

E-Cadherin Is Important for Meibomian Gland Function as Revealed by a New Human ex Vivo Slice Culture Model

“…Recent attempts of establishing a suitable in vitro three-dimensional meibomian culture model also faced limitations. 11 Together with the lack of appropriate animal models, 11,12 we are aware of the urgent requirement of an appropriate model system to study the (patho-)physiology of meibomian glands. Therefore, recently established ex vivo slice culture models were adapted to study cell cohesion in human skin and murine heart 27,38 and established a novel ex vivo slice culture model of human eyelids.…”

“…However, at the moment, many in vitro studies are confronted with limitations in reflecting the in vivo situation. 4,10,11 Moreover, up to now no satisfactory animal model exists that resembles the pathophysiologic aspects of human MGD. 11,12 Together, this underlines the requirement of new model systems for the investigation of the (patho-)physiology of meibomian glands in a physiologic environment.…”