Abstract
Purpose :
Oral mucosal stem cells are the key cells of rapidly dividing cells in buccal tissues. This easily accessible and on-demand cells can multiple and regenerate damaged epithelium and may have the ability to differentiate into corneal epithelial cell type. The aim of the study was to isolate cells from a buccal biopsy and expand them under clinical grade condition for use in future translational applications to reconstruct the ocular surface.
Methods :
A biopsy of rabbit buccal tissue was used and cells were isolated and seeded on a cGMP-certified cell culture surface. Cells were expanded with a newly designed clinical-grade xeno-free medium with no feeder. Collagenase treatment was used to detach and harvest the cell sheet. Live cells imaging and morphological analysis were used under clinical grade conditions to examine cell growth, cell morphology and cell sheet production. Western blot analysis was used to detect levels of stem cell marker expression, as well as corneal epithelial cell markers.
Results :
Cells attached on to the surface and self-assembled into colony forming units (CFUs). Microscopic examination showed that these CFUs formed during the first 5 days, and that a monolayer cell sheet formed in less than 10 days. Cells differentiated and a multilayered epithelial cell sheet was harvested after 17 days. Immunostaining analysis showed that DeltaNp63 was expressed in the basal cells of the cell sheet. Western blot analysis showed a positive detection of Bmi-1 and Pax-6, indicating the expression of stem cells markers in the cell sheet. K3/K12 positive expression indicated that the produced cell sheet was differentiated into corneal epithelial cells-like. Collagenase treatment, used for detaching and harvesting the cell sheet, did not affect focal adhesion molecules (i.e., INTGB1, 4 and phosphorylated FAK) as well as adhesion molecules (such as E-cadherin and beta-catenin).
Conclusions :
The present study reports the successful expansion of rabbit oral mucosal cells in a newly designed clinical-grade xeno-free medium with no feeder. Their differentiation into corneal epithelial-like cells and expansion as cell sheets could be used in the future to reconstruct the ocular surface of patients with limbal stem cell deficiency.
This is a 2020 ARVO Annual Meeting abstract.