A novel, simple, and reproducible method for cultivating pathological tissues obtained from human eyes during surgery was developed using viscoelastic material as a tissue adherent to facilitate cell attachment and expansion and calcium imaging of cultured cells challenged by mechanical and acetylcholine (ACh) stimulation as well as inflammatory studies. Anterior lens capsule-lens epithelial cells (aLC-LECs) from cataract surgery and proliferative diabetic retinopathy (PDR) fibrovascular epiretinal membranes (fvERMs) from human eyes were used in the study. We hereby show calcium signaling in aLC-LECs by mechanical and acetylcholine (ACh) stimulation and indicate presence of ACh receptors in these cells. Furthermore, anex vivostudy model was established for measuring the inflammatory response in fvERMs and aLC-LECs upon TNFα treatment.

Cataract is the leading cause of blindness worldwide but the mechanisms involved in the process of cataractogenesis are not yet fully understood. Two most prevalent types of age-related cataracts are nuclear (N) and cortical (C) cataracts. A common environmental factor in most age-related cataracts is believed to be oxidative stress. The lens epithelium, the first physical and biological barrier in the lens, is build from lens epithelial cells (LECs). LECs are important for the maintenance of lens transparency as they control energy production, antioxidative mechanisms and biochemical transport for the whole lens. The purpose of this study is to characterize compounds in LECs originated from N and C cataracts, by using the synchrotron radiation-based Fourier Transform Infrared (SR-FTIR) microspectroscopy, in order to understand the functional importance of their different bio-macromolecules in cataractogenesis. We used the SR-FTIR microspectroscopy setup installed on the beamline MIRAS at the Spanish synchrotron light source ALBA, where measurements were set to achieve single cell resolution, with high spectral stability and high photon flux. The results showed that protein aggregation in form of fibrils was notably pronounced in LECs of N cataracts, while oxidative stress and the lipids peroxidation were more pronounced in LECs of C cataracts.

In this work we investigated how modifications of the Ca²⁺ homeostasis in anterior lens epithelial cells (LECs) are associated with different types of cataract (cortical or nuclear) and how the progression of the cataract (mild or moderate) affects the Ca²⁺ signaling. We systematically analyzed different aspects of intra- and inter-cellular Ca²⁺ signaling in the human LECs, which are attached to surgically isolated lens capsule (LC), obtained during cataract surgery. We monitored the temporal and spatial changes in intracellular Ca²⁺ concentration after stimulation with acetylcholine by means of Fura-2 fluorescence captured with an inverted microscope. In our analysis we compared the features of Ca²⁺ signals in individual cells, synchronized activations, spatio-temporal grouping and the nature of intercellular communication between LECs. The latter was assessed by using the methodologies of the complex network theory. Our results point out that at the level of individual cells there are no significant differences when comparing the features of the signals with regard either to the type or the stage of the cataract. On the other hand, noticeable differences are observed at the multicellular level, despite inter-capsule variability. LCs associated with more developed cataracts were found to exhibit a slower collective response to stimulation, a less pronounced spatio-temporal clustering of LECs with similar signaling characteristics. The reconstructed intercellular networks were found to be sparser and more segregated than in LCs associated with mild cataracts. Moreover, we show that spontaneously active LECs often operate in localized groups with quite well aligned Ca²⁺ activity. The presence of spontaneous activity was also found to affect the stimulated Ca²⁺ responses of individual cells. Our findings indicate that the cataract progression entails the impairment of intercellular signaling thereby suggesting the functional importance of altered Ca²⁺ signaling of LECs in cataractogenesis.

Characterization of the cell surface marker phenotype ofex vivocultured cells growing out of human fibrovascular epiretinal membranes (fvERMs) from proliferative diabetic retinopathy (PDR) can give insight into their function in immunity, angiogenesis, and retinal detachment. FvERMs from uneventful vitrectomies due to PDR were cultured adherentlyex vivo. Surface marker analysis, release of immunity- and angiogenesis-pathway-related factors upon TNFαactivation and measurement of the intracellular calcium dynamics upon mechano-stimulation using fluorescent dye Fura-2 were all performed. FvERMs formed proliferating cell monolayers when culturedex vivo, which were negative for endothelial cell markers (CD31, VEGFR2), partially positive for hematopoietic- (CD34, CD47) and mesenchymal stem cell markers (CD73, CD90/Thy-1, and PDGFRβ), and negative for CD105. CD146/MCAM and CD166/ALCAM, previously unreported in cells from fvERMs, were also expressed. Secretion of 11 angiogenesis-related factors (DPPIV/CD26, EG-VEGF/PK1, ET-1, IGFBP-2 and 3, IL-8/CXCL8, MCP-1/CCL2, MMP-9, PTX3/TSG-14, Serpin E1/PAI-1, Serpin F1/PEDF, TIMP-1, and TSP-1) were detected upon TNFαactivation of fvERM cells. Mechano-stimulation of these cells induced intracellular calcium propagation representing functional viability and role of these cells in tractional retinal detachment, thus serving as a model for studying tractional forces present in fvERMs in PDRex vivo.