Ex - vivo cultivation of limbal epithelial cells and corneal stromal cells on electrospun plasma treated poly-ε-caprolactone scaffolds for ocular surface reconstruction

Abstract

Purpose: To evaluate viability of co-cultured limbal epithelial cells and corneal stromal cells on a synthetic substrate that can serve as an alternative strategy for corneal tissue bio engineering.

Methods: Human corneal tissues not suitable for transplantation were used in the study. Limbal epithelial cells were isolated using Trypsin EDTA (0.25%) and corneal stromal cells were expanded by explant culture method in separate 35mm petri dishes. Cells were cultured in culture media DMEM/Ham’s F12 (3:1), fetal bovine serum (10%) with growth factors. Nanofibers were fabricated using 10 wt% poly-ε-caprolactone (PCL), solution dissolved in 2, 2, 2-trifluoroethanol by electrospinning process and plasma treatment was carried out using Oxygen-Helium plasma. Nanofibers were characterized for surface morphology, wetting ability, pore size, and mechanical strength. For co-culture, 10000 cells of each cell type were seeded on plasma PCL, human amniotic membrane (HAM) and glass cover slip for 14 days. Parameters evaluated included cellular phenotypic profile, viability, proliferation, and attachment ability. Bio compatibility, surface morphology and proliferation of cells on nanofibers were observed on day 3, 5, 7 and 14 days. Immunocytochemistry and RT-PCR was done to check the characteristics molecular markers for limbal epithelial and corneal stromal cells.

Results: Cell-viability staining results indicated that the nanofiber surface was biocompatible for co-cultured cells. SEM images showed that co-cultured cells formed a continuous cell sheet on nanofibers. The proliferative capacity of co-cultured cells on plasma PCL was less then cells grown on HAM and glass cover slip but data was not statistically significant (p value> 0.05). RT-PCR and Immunocytochemistry results showed no change in the expression profile of LEC’s for Cytokeratin-3, ABCG2, p63, Beta-one-Integrin and corneal stromal cells markers Vimentin, Keratocan, Aldehyde-3- dehydrogenase and smooth muscle actin grown on plasma-PCL compared to cells grown on cover slips and human amniotic membrane.

Conclusions: Plasma treated PCL supported expansion of corneal co-cultured cells; therefore it can serve as an useful alternative carrier for ocular surface tissue engineering. Co-cultured cells may be useful in management to support wound healing in corneal and scleral ulceration.

Keywords: 482 cornea: epithelium • 484 cornea: stroma and keratocytes