Prostaglandin F2α Agonists Negatively Modulate the Size of 3D Organoids from Primary Human Orbital Fibroblasts

Abstract

To elucidate the molecular etiology of deepening of the upper eyelid sulcus (DUES) induced by prostaglandin (PG) analogs, a three-dimensional (3D) tissue culture system was employed using human orbital fibroblasts (HOFs). METHODS. During adipogenesis, changes in HOF 3D organoid sizes, as well as their lipids stained by BODIPY and expression of the extracellular matrix (ECM) by immunolabeling and/or quantitative PCR, were studied in the presence or absence of either 100-nM bimatoprost acid or 100-nM prostaglandin F2α. RESULTS. The size of the 3D organoids increased remarkably during adipogenesis, but such increases were significantly inhibited by the presence of PG analogs. Staining intensities by BODIPY and mRNA expression of peroxisome proliferator-activated receptor gamma were significantly increased upon adipogenesis but were not influenced by the presence of PG analogs. Unique changes in ECM expression observed with or without adipogenic differentiation were significantly modified by the presence of PG analogs. CONCLUSIONS. Our present study indicates that PG analogs have the potential to modulate the ECM network within HOF 3D organoids. Thus, a 3D tissue culture system may be a suitable strategy for understanding the disease etiology of DUES.

ROCK inhibitors beneficially alter the spatial configuration of TGFβ2-treated 3D organoids from a human trabecular meshwork (HTM)

“…Furthermore, these 3D culture techniques may have some limitations in terms of mimicking the physiological and pathological conditions because of the presence of an unnecessary scaffolds. Alternatively, our group recently developed a 3D cell drop culture system in which no scaffold is required 25 – 27 . Using this technique, we were able to obtain 3D organoids from HOF in patients with TAO using a drop culture method, and found that HIF2A played a critical role in mediating LOX-dependent ECM accumulation 25 .…”

“…Using this technique, we were able to obtain 3D organoids from HOF in patients with TAO using a drop culture method, and found that HIF2A played a critical role in mediating LOX-dependent ECM accumulation 25 . More recently, to elucidate the molecular etiology of DUES induced by PGs, we employed this 3D culture system using HOF and 3T3-L1 cells, and found that PGs significantly suppressed 3D organoid sizes, and modulated the spatial distribution of several ECMs surrounding the 3D organoids 26 , 27 . We therefore concluded that our developed 3D cell culture would be suitable for investigating the molecular etiology of TAO and DUES and also will be applicable for use in many other cells and organs.…”

“…Immunocytochemistry of 3D HTM organoids was conducted by the method described recently 26 , 27 . All procedures were performed at RT unless otherwise stated.…”

“…Although most of these studies involved the use of conventional 2D cell cultures, despite the fact that the HTM is composed of multiple sheets in the human eye 24 , thus a relevant 3D cell culture model should be used in this research field. Our group recently established a 3D cell culture system using 3T3-L1 cells or human orbital fibroblasts (HOF) as disease models for thyroid associated orbitopathy (TAO) 25 , and the deepening of the upper eyelid sulcus (DUES) induced by prostaglandin analogues (PGs) 26 , 27 .…”

Omidenepag, a non-prostanoid EP2 receptor agonist, induces enlargement of the 3D organoid of 3T3-L1 cells

“…In fact, quite recently, such 3D tissue cultures have been used in conjunction with several human disease models 14 . Using the 3D organoid culture technique, in our previous studies, we found that PGF2α significantly affected adipogenesis and the extracellular matrixes (ECMs) in 3T3-L1 cells 16 as well as human orbital fibrobrasts (HOFs) 17 . Therefore, in the current study, using a replicated pathogenic DUES model by 2D and 3D cultures using 3T3-L1 cells, we analyzed and compared the effects of FP and EP agonists, OMD and butaprost (Buta), on adipogenesis, ECM expression, and the sizes and physical properties of the 3D organoids.…”