Abstract
Purpose :
Ocular surface diseases (OSD) in severe forms as seen in such cicatricial conditions, chemical burns, limbal cell deficiency, and neurotrophic disease, still impose therapeutic challenges and vision threats. In the precision medicine field, limbal stem cells (LSC) are a promising tool for cell therapy and ocular surface regeneration. There is no consensus about LSC isolation and tissue sources are limited, but the donor scleral-corneal ring is usually discarded after corneal transplantation. Thus, we aimed to standardize a protocol of LSC isolation from donor scleral-corneal tissues for future pre-clinical and clinical applications.
Methods :
Five donor scleral-corneal rings were collected after corneal transplantation in partnership with the Biobank of the Brazilian Biosciences National Laboratory (LNBio/CNPEM) from Campinas, SP, Brazil. Several protocols of decontamination were tested, followed by microbiological control analyses. We also tested two different enzymatic digestion protocols to isolate LSC, using Dispase or Collagenase I. For validation, cell line authentication was performed by Short Tandem Repeats (STR) analyses, besides cell counting, and morphological evaluation.
Results :
As standard and validated protocol, the limbic area of the scleral-corneal rings was cut into small fragments (3x3mm), washed with PBS, and incubated overnight with antibiotic/antimycotic solution for decontamination. For isolation of LSC cells, fragments were incubated overnight with Collagenase I in DMEM at 37Celcius degree. The remaining tissue was dissociated, filtered, and centrifuged. The pellet of cells was resuspended, counted, and cultured or frozen. The cellular identity was confirmed by STR analysis. Cell counting demonstrated a viability of cell population over 98% since passage 0. The population of isolated LSC presented fibroblastoid morphology, typical of mesenchymal stem cells.
Conclusions :
LSC can naturally regenerate the ocular surface and is a promising tool for composing new advanced therapy medicinal products to treat OSD. Considering that the sources for this cell type are limited, we highlight the importance of a proper method of LSC isolation using donor scleral-corneal rings after corneal transplantation. The application of the present protocol will allow an increase in the number of LSC available for pre-clinical and clinical studies progress.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.