Ex vivo adherent cultures for ocular disease modeling, characterisation and inflammation studies

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Development of ex vivo models to study pathogenesis, inflammation and treatment modalities for different eye disorders such as pterygium, cataract and fibrovascular epiretinal membranes (fvERMs) from proliferative diabetic retinopathy (PDR) is much needed. A novel, simple, and reproducible method for cultivating such pathological tissues obtained from human eyes during surgery was hereby developed using viscoelastic material as a tissue adherent to facilitate cell attachment. Pterygium obtained from surgery was cultivated until reaching 3D stratification over long periods of cultivation ex vivo (3 months). Surface profiling of the multi-layered outgrowing cells from the pterygium was performed to test for presence of hematopoietic- and mesenchymal- stem cell markers, as well as determine the origin of the outgrowing cells. Examination of these cells by immunohistochemistry using pluripotency, oxidative stress, stemness, migration and proliferation, epithelial and secretory markers was also performed, while the effect of anti-proliferative agent Mitomycin C upon secretion of pro-inflammatory cytokines IL-6 and IL-8 was assessed. The 3D outgrowing cells from the pterygium showed high expression of migration- (CXCR4), secretory- (MUC1, MUC4) and oxidative damage- (8-OHdG) markers, and low expression of hypoxia- (HIF-1α) and proliferation- (Ki-67) markers. Moderate and low expression of the pluripotency markers (Vimentin and ΔNp63) was present, respectively, while the putative markers of stemness (Sox2, Oct4, ABCG-2) and epithelial cell markers- (CK19, CK8-18) were weakly expressed. The surface marker profile of the outgrowing cells revealed high expression of the hematopoietic marker CD47, mesenchymal markers CD90 and CD73, minor or less positivity for the hematopoietic marker CD34, mesenchymal marker CD105, progenitor marker CD117 and attachment protein markers. The 3D outgrowing cells secreted low levels of IL-6 and IL-8 ex vivo, which were inhibited upon Mitomycin C treatment. The ex vivo tissue engineered pterygium consists of a mixture of cells of different lineage origin, suitable for use as a disease model for studying pathogenesis ex vivo, while opening possibilities for new treatment and prevention modalities. Furthermore, anterior lens capsule-lens epithelial cells (aLC-LECs) from cataract surgery and fvERMs from human eyes undergoing vitrectomy for PDR were used. The aLC-LECs and the fvERM cells were expanded ex vivo in a 2D-monolayer culture. The aLC-LECs underwent calcium imaging upon challenge by mechanical and acetylcholine (ACh) stimulation, while the fvERM outgrowing cells underwent inflammatory studies. The aLC-LECs stimulated by mechanical and ACh treatment showed active calcium signaling and presence of ACh receptors in these cells. The fvERMs could be activated to undergo an inflammatory response upon TNFα treatment, which can surve as an ex vivo study model for studying PDR and inflammation in vivo –like conditions.

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