The MGs are located within the eyelid and are responsible for synthesizing and secreting lipids that constitute the primary source of the tear film's lipid layer. This lipid layer is essential for preserving the stability of the tear film and maintaining ocular surface homeostasis [1]. It is generally believed that duct obstruction of the MGs or qualitative and quantitative changes in gland secretion may lead to tear film disruption, ocular irritation symptoms, including clinical inflammation, gland atrophy and other related complications, which are referred to as MGD [2]. MGD is a prevalent cause of ocular surface diseases, including dry eye [3]. To investigate the pathogenesis and underlying mechanisms of ocular injury associated with MGD, an ex vivo culture system of MG tissue or cells is essential and indispensable.

The immortalized human meibomian gland epithelial cell line, established in 2010 [4], has been extensively utilized to assess the impact of hormones [5], serum [4], and pharmaceuticals [6] on meibomian gland cells. This cell line is a valuable tool for investigating the physiology and pathology of MGs. However, the conventional 2D culture system is inadequate in replicating the natural microenvironment of MGs in vivo, which involves intricate interactions with fibroblasts, melanocytes, immune cells [7], and others. The ex vivo culture of tissues and organs can offer a 3D microenvironment for target cells, enabling researchers to replicate the native conditions more faithfully and derive more precise conclusions [8]. Consequently, an increasing number of research teams have attempted to employ isolated MGs for direct organotypic culture.

Rötzer et al. embedded isolated living MG tissue in low-melting point agarose, and subsequently sliced the tissue into 300 μm thick slices using a vibrating microtome after solidification for culture purposes [9]. This method of thick tissue sectioning preserves the integrity of the MG and surrounding tissue, enabling the investigation of the interactions between MG acinar cells and their microenvironment. Zahn et al. adapted the culture system by adjusting tissue section thickness to 150 μm, aiming to sustain ex vivo MG viability for up to 21 days [10]. However, the aforementioned method involves invasive sectioning, which may potentially trigger a stress response from meibomian cells surrounding the wounded area and thus impact research outcomes. Xu et al. developed an alternative method for culturing MGs, utilizing intact glands instead of sectioned tissue [11]. Tarsal plates were immobilized with the matrigel before adding to immerse the MG for cultivation. Regrettably, this culture system was only able to sustain tissue viability for a limited duration, exhibiting a decline of over 40% in viability after five days of cultivation. The decline in vitality could potentially be attributed to inadequate oxygen supply due to submerged culture. In addition, the media employed in the three aforementioned studies were either DKSFM or DMEM/F12 supplemented with 10% serum, neither of which has been validated to support the rapid and consistent proliferation of primary meibomian gland epithelial cells in vitro [12]. The utilization of inappropriate media may potentially alter the gene expression profile of meibomian gland cells and result in unintended differentiation [13].

In this study, we developed an ex vivo organotypic culture model for MGs that supports the growth and viability of meibomian gland cells. The effectiveness of this new model was verified by utilizing MGs from mice of different ages and subjected to drug treatments. This model has broad applications in investigating the pathophysiology of MGD and screening of drugs for MGD treatment.