Corneal Cell Biology
In this WG research will be combined to study the characteristics of the corneal cells that have been cultured on the biological scaffolds developed in WP1. Cornealized scaffolds produced using the optimized culture protocols (developed in WP2) need to be validated. The aim is to show that the cells cultured on these bio-scaffolds, using the developed culture protocols, are as similar to in vivo conditions as possible. Exchange of cell growth experience is absolutely essential.
This WG has three research objectives which depend on the collaboration of different laboratories and exchange of experience, namely:
1. To determine the phenotypic characteristics of the cultured cells;
2. To study the cell-scaffold interactions;
3. To study the secreted proteins in the extracellular milieu
Research tasks to achieve these objectives are:
• Task 3.1: To determine the phenotypic characteristics of the cultured cells. Scanning electron microscopy (SEM) will be carried out to examine apical surface cellular structures (e.g. microvilli, microplicae etc). TEM will be done to examine cellular arrangement, stratification, ultra structure of the apical, wing and basal epithelial cells as well as cell-cell anchoring structures (desmosomes, anchoring fibrils and plaques). Labelling with various antibodies against membranous, cytoplasmic and nuclear proteins will be carried out to determine characteristics of corneal cell phenotype. The Action’s website will be used to share experiences and techniques, leading to a series of standard-operating procedures.
• Task 3.2: To study the cell-scaffold interactions: TEM will be performed to determine presence of cell-scaffold anchoring ultra-structures such as hemidesmosomes, basal lamina, anchoring fibrils and plaques. Labelling with antibodies against various basement membrane and extracellular matrix (ECM) proteins will be done to ascertain presence of basement membrane and ECM components. The knowledge will be combined in a database on the basic necessities of testing new scaffolds.
• Task 3.3: To study the secreted proteins in the extracellular milieu. Culture supernatants will be collected and analysed for the presence of secreted proteins. This will be performed by cytometric bead arrays and/or Enzyme-Linked Immuno Sorbent Assay (ELISA), luminex assays and flow cytometry. Various culture protocols (from WP2) will be tested for inflammatory capacity by detecting presence of inflammatory cytokines (IL-1, IL-6, IL-8, TNFα, etc.).