Description
The overall objective of this proposal is to help to alleviate the dual problem facing transplantation medicine in ophthalmosurgery: shortage of donor tissues and its relatively short storage time. We will develop novel improved protocols for preparation of corneal (endothelial and epithelial) and conjunctival (epithelial and goblet) cells for potential grafting in human medicine. The project promoter, and its partners, will work together on following workpackages: reparation and self-regeneration of the corneal endothelium, preparation of endothelial, limbal and conjunctival cells for grafting, testing DNA integrity of cells for grafting. The benefits consist in developing of methods and procedures which will lead to introduction of transplantation methods not yet performed in the Czech Republic. Moreover, using the vitrification technique we aim to develop protocols to long-term storage of cells/tissues intended for direct grafting or for generating transplantable grafts. The control of DNA integrity in cultured cells and tissues will be a substantial contribution in terms of safety of transplantation.
Summary of project results
The main outcomes are the introduction of the preparation of limbal stem cells in laboratory conditions at the workplace of the promoter and the finding of the most suitable experimental conditions in terms of cell stemness preservation and certification process in the frame of Czech Republic health legislation (Manuscript, Manu 1). We identified a new protein in cornea - inhibitor of proteinase 9. We determined its localization in all layers of the cornea, limbus and conjunctiva. Experimentally, we confirmed the PI-9 function in corneal pathology – it is involved in inflammation and graft rejection after corneal transplantation (Sub 5). At the cell-molecular level, we also characterized the pathological endothelium and contributed to elucidating the pathogenesis of endothelial (Paper 2, Paper 5) and stromal dystrophies (Paper 10), limbal stem cell deficiency (Manu 6) and pterygium (Paper 8). We have published new treatments procedures using amniotic membrane and autologous serum (Paper 1, Paper 7). We have determined the effect of endothelial quality on the preparation of the lamellar graft (Sub 1). A two-scale corneal repair/regeneration model was developed, and the results show that the corneal tissues containing the area beyond their periphery repair more efficiently than corneas without this area (Manu 7). We found that conjunctiva reflects genotoxic damage to the eye surface (Manu 8), we have applied new genotoxic tests to cells of the eye surface (Sub 2, Manu 9). We standardized the conditions for the cultivation of goblet cells from limbal area, but we did not succeed to stimulate their proliferation (Manu 10). We developed a new technique of cell cultures cryopreservation using vitrification without cryoprotectants (Manu 11) and verified the stemness preservation of limbal cells preserved by this method. (Manu 12). In addition to deepening of our knowledge, which will lead directly to the introduction of new clinical methods closely related to corneal cell transplantation, the project had an important socio-economic impact; it helped professional reincorporation of three women after their maternity leave.
Summary of bilateral results
The donor project partner contributed to the project at a technical/professional level so a limbal stem cell cultivation /comet assay application for eye cells introduced at partner/donor side The partnership contribute to the project outcome and outputs with experience sharing and joint publications. The partnership achieved concerning strengthened bilateral relations experience sharing, internationally refereed joint publications with high impacts, extended, more sharply directed research. What are the socio-economic impact and the wider societal implications of the project, including gender equality actions, ethical issues, and wider awareness? The project supported 21 female researchers, 3 reincorporated after maternity leave, 6 Ph.D. students, 21 postdocs involved. In addition to deepening of our knowledge, which will lead directly to the introduction of new clinical methods closely related to corneal cell transplantation, the project had an important socio-economic impact; it helped professional reincorporation of three women after their maternity leave.