Isolation, characterization and production of limbal stem cells for application on eye surface diseases

Abstract

New techniques and methodologies are being developed for the treatment of ocular surface diseases (OSD), such as dry eye, limbal stem cell deficiency, and neurotrophic disease. Among the existing techniques, some of the most promising ones are related to the use of stem cells in cellular therapy and regenerative medicine. In this context, limbal stem cells (LSCs) represent a cellular ensemble present at the transition between the cornea and the sclera (corneoscleral junction) and act in the maintenance and proper differentiation of the corneal epithelium, as they demonstrate a degree of undifferentiation, as well as the capacity for self-renewal. These cells have already been used in the treatment of ocular burns, however, there is no consensus regarding their therapeutic use, requiring further studies in the field. This way, this project aims to standardize the isolation, cultivation, characterization, and cryopreservation of LSCs for future pre-clinical and clinical applications. For this purpose, at least 5 samples of corneoscleral rings will be provided by the biobank of the Brazilian Biosciences National Laboratory from the Brazilian Center for Research in Energy and Materials (LNBio/CNPEM - CONEP number: B-111). Different cellular processing protocols will be tested, along with the characterization of LSCs regarding morphology, duplication rate and senescence, immunophenotypic profile, tri-lineage differentiation capacity, and gene expression pattern. All stages will undergo microbiological and quality control to ensure an experimental design with potential clinical application, as LSCs may act in corneal tissue regeneration and have great potential as a cellular therapy product in the treatment of OSD.