Purpose. To develop a complete and optimized method to expand in culture human conjunctival epithelial cells from cadaveric donor samples. Methods. Epithelial cells were obtained from cadaveric conjunctival tissue (n = 47). Preplating and differential trypsinization were optimized to eliminate stromal contamination. Epithelial cells were grown with five different media: control, epithelial growth factor (EGF)-enriched, H₂O₂-supplemented, fibroblast-conditioned, and human serum media. Adhesion, proliferation, colony forming efficiency (CFE), and percentage of CK19⁺ and Ki67⁺ cells were determined with the five different media. Cells were characterized by immunofluorescence and/or Western blotting techniques for the expression of CK4, CK7, CK19, MUC5AC, vimentin, FSP-1, Ki67, E-cadherin, and zonula occludens (ZO)-1 markers. In addition, cells were treated with TNF-α and levels of secreted IL-6 were measured by enzyme-linked immunosorbent assay. Results. Pure epithelial cell cultures were obtained. Human serum medium showed the best properties in proliferation and CFE, while maintaining epithelial phenotype. Cells with this medium were passaged up to five times, although they maintained all epithelial characteristics only through passage 3. Cultured cells expressed epithelial markers, but not stromal ones. The number of MUC5AC⁺ cells increased throughout the passages, whereas Ki67⁺ cell numbers decreased. Cells in culture maintained adherens and tight junctions, and responded to TNF-α treatment by releasing more IL-6, showing that they can be used for inflammation assays. Conclusions. We have developed a complete protocol to expand conjunctival epithelial cells from cadaveric tissue. This culture system responded to an inflammatory stimulus, so it could be used to develop a more complex in vitro model of inflammation.

This review focuses on conjunctival goblet cells and their essential function in the maintenance of eye health. The main function of goblet cells is to produce and secrete mucins that lubricate the ocular surface. An excess or a defect in those mucins leads to several alterations that makes goblet cells central players in maintaining the proper mucin balance and ensuring the correct function of ocular surface tissues. A typical pathology that occurs with mucous deficiency is dry eye disease, whereas the classical example of mucous hyperproduction is allergic conjunctivitis. In this review, we analyze how goblet cell number and function can be altered in these diseases and in contact lens (CL) wearers. We found that most published studies focused exclusively on the goblet cell number. However, recent advances have demonstrated that, along with mucin secretion, goblet cells are also able to secrete cytokines and respond to them. We describe the effect of different cytokines on goblet cell proliferation and secretion. We conclude that it is important to further explore the effect of CL wear and cytokines on conjunctival goblet cell function.

The aim of this study was to develop a three-dimensional model of the human conjunctiva that can be used to perform physiology and pathophysiology experiments. Fibrin-based matrices (derived from human plasma or plasma cryoprecipitate) were used as scaffolds, and primary cells were obtained from conjunctival tissue. Conjunctival constructs were analyzed by immunofluorescent staining and scanning electron microscopy and cell proliferation was measured with alamarBlue^® assay. After characterizing the constructs, four different experimental conditions were analyzed in cryoprecipitate matrices: controls, air-lifted cultures (to increase cell stratification), partially desiccated cultures (to mimic dry eye disease), and IL-13-treated cultures (to mimic allergy). Constructs were stained with hematoxylin/eosin to observe changes in morphology. High molecular weight glycoconjugates were identified by HPA staining. MUC5AC and IL-6 secretion was evaluated by ELISA. The fibrin-based matrices supported conjunctival cell growth. Epithelial cells grew on the surface of the scaffolds and underwent stratification that increased over time. These cells had microvilli, which suggests cell polarization and functionality. Fibroblasts were integrated in the scaffold and showed elongated shape. Compared to controls, air-lifted construct had increased epithelial stratification and upregulated MUC5AC secretion. Increased MUC5AC secretion also occurred in partially desiccated and IL-13-treated cultures. The inflammatory status of cells was evaluated by IL-6 levels which were increased in air-lifted and partially desiccated cultures, but not in IL-13-treated ones. In conclusion, we have developed a new three-dimensional model of human conjunctiva that can be used to study ocular surface inflammatory diseases.

Purpose: Increased expression of transforming growth factor-β2 (TGF-β2) is reported in the conjunctiva of dry eye patients with no increase of anti-inflammatory activity of TGF-β2. Our aim was to compare the expression of molecules involved in TGF-β2 activation, thrombospondin-1 (TSP-1) and CD36, during murine and human conjunctival inflammation. Methods: Human conjunctival tissue from cadaveric donors, human conjunctival epithelial primary cells and fibroblasts, and murine conjunctivas were immunostained for TSP-1, CD36, or TGF-β2. Inflamed conjunctival tissues were obtained from C57BL/6 wild-type (WT) mice induced to develop experimental dry eye (EDE) with 10 days of desiccating conditions and scopolamine injections and TSP-1-deficient (TSP1^−/−) mice, which spontaneously develop Sjögren's syndrome-associated conjunctival inflammation with age. Immunostaining intensities were compared using ImageJ software. Cultures of human conjunctival fibroblasts were stimulated with IL-1β and both secreted protein and message levels of TSP-1, CD36, and TGF-β2 were analyzed. Results: TSP-1 and CD36 were detectable in human and murine conjunctival tissues as well as primary conjunctival epithelial cells and fibroblasts. Increased conjunctival immunostaining of TGF-β2 and reduced CD36 were detected in EDE mice compared with WT mice. Interestingly, increased TGF-β2 and CD36 conjunctival immunostaining was detected in TSP1^−/− mice. The expression of TSP-1 and CD36 was downregulated in IL-1β-stimulated conjunctival fibroblasts at both the protein and message level, while active TGF-β2 was undetected. Conclusions: The absence or reduced expression of either of the molecules involved in TGF-β2 activation supports proinflammatory conditions in the conjunctiva. Changes in TSP-1 and CD36 may serve as potential biomarkers of conjunctival inflammation.

Proteomics is a crucial tool for unravelling the molecular dynamics of essential biological processes, becoming a pivotal technique for basic and applied research. Diverse bioinformatic tools are required to manage and explore the huge amount of information obtained from a single proteomics experiment. Thus, functional annotation and protein–protein interactions are evaluated in depth leading to the biological conclusions that best fit the proteomic response in the system under study. To gain insight into potential applications of the identified proteins, a novel approach named “Applied Proteomics” has been developed by comparing the obtained protein information with the existing patents database. The development of massive sequencing technology and mass spectrometry (MS/MS) improvements has allowed the application of proteomics nonmodel microorganisms, which have been deeply described as a novel source of metabolites. Between them, Nannochloropsis gaditana has been pointed out as an alternative source of biomolecules. Recently, our research group has reported the first complete proteome analysis of this microalga, which was analysed using the applied proteomics concept with the identification of 488 proteins with potential industrial applications. To validate our approach, we selected the UCA01 protein from the prohibitin family. The recombinant version of this protein showed antiproliferative activity against two tumor cell lines, Caco2 (colon adenocarcinoma) and HepG-2 (hepatocellular carcinoma), proving that proteome data have been transformed into relevant biotechnological information. From Nannochloropsis gaditana has been developed a new tool against cancer—the protein named UCA01. This protein has selective effects inhibiting the growth of tumor cells, but does not show any effect on control cells. This approach describes the first practical approach to transform proteome information in a potential industrial application, named “applied proteomics”. It is based on a novel bioalgorithm, which is able to identify proteins with potential industrial applications. From hundreds of proteins described in the proteome of N. gaditana, the bioalgorithm identified over 400 proteins with potential uses; one of them was selected as UCA01, “in vitro” and its potential was demonstrated against cancer. This approach has great potential, but the applications are potentially numerous and undefined.