Preserving Basement Membranes during Detachment of Cultivated Oral Mucosal Epithelial Cell Sheets for the Treatment of Total Bilateral Limbal Stem Cell Deficiency

Abstract

Total bilateral limbal stem cell deficiency leading to loss of corneal clarity, potential vision loss, pain, photophobia, and keratoplasty failure cannot be treated by autologous limbal transplantation, and allogeneic limbal transplantation requires subsequent immunosuppressive treatment. Cultured autologous oral mucosal epithelial cells have been shown to be safe and effective alternatives. These cells can be transplanted on supports or without support after detachment from the culture dishes. Dispase, known for epidermal sheet detachment, is reported as not usable for oral mucosa. The objective was to find an optimized detachment method providing a sufficiently resistant and adhesive cultured oral mucosal epithelium (COME), which can be grafted without sutures. Enzymatic treatments (dispase or collagenase at different concentrations) were compared to enzyme-free mechanical detachment. Histological immunofluorescence (IF) and Western blotting (WB) were used to examine the impact on adhesion markers (laminin-332, β1-integrin, and type VII collagen) and junctional markers (E-cadherin, P-cadherin). Finally, the COME ability to adhere to the cornea and produce a differentiated epithelium 15 d after grafting onto an ex vivo porcine stroma model were investigated by histology, IF, and transmission electron microscopy. Collagenase at 0.5 mg/mL and dispase at 5 mg/mL were selected for comparative study on adhesive expression marker by IF and WB showed that levels of basement membrane proteins and cell–cell and cell–matrix junction proteins were not significantly different between the 3 detachment methods. Collagenase 0.5 mg/mL was selected for the next step validation because of the better reproducibility, 100% success (vs. 33% with dispase 5 mg/mL). Grafted onto porcine de-epithelialized corneal stroma, collagenase 0.5 mg/mL detached COME were found to adhere, stratify, and continue to ensure renewal of the epithelium. For COME, collagenase 0.5 mg/mL enzymatic detachment was selected and validated on its resistance and adhesive marker expression as well as their anchorage onto our new ex vivo de-epithelialized stroma model.