Enhancing Ex Vivo Limbal Epithelial Cell Expansion on Amniotic Membrane: A Comparative Study of Monolayer (2D) Versus Sandwich (3D) Culture Configurations

Abstract

Purpose: This study compared two-dimensional (monolayer) and three-dimensional (sandwich) systems for expanding ex vivo limbal epithelial cells on amniotic membrane and evaluated the outcomes after transplantation into rabbits with experimentally induced limbal stem cell deficiency. Methods: Evaluations included markers for progenitor cells, proliferation, apoptosis, and clinical monitoring for up to 63 days. In the monolayer culture, epithelial cells derived from limbal explants were expanded on amniotic membrane as the substrate. In the sandwich culture, the cells were cultured between 2 layers of amniotic membrane. Evaluations included markers for progenitor cells, proliferation, and apoptosis, along with clinical monitoring for up to 63 days. Results: Sandwich cultures demonstrated increased cellular proliferation and fewer progenitor cells compared with monolayer cultures. In treating limbal stem cell deficiency, the group receiving transplantation from sandwich cultures exhibited reduced neovascularization and decreased corneal ulceration compared with those treated with monolayer cultures, with similar clinical outcomes in corneal opacity. The configuration of the culture system did not affect the presence of apoptotic cells. Corneas treated with sandwich cultures showed a higher presence of progenitor cells compared with the monolayer group, suggesting a potential long-term viability advantage for these transplants. Conclusions: In conclusion, although the sandwich culture system enhanced cellular proliferation, it also resulted in a decrease in progenitor cells within the cultures. Nevertheless, both systems demonstrated comparable therapeutic efficacy in treating limbal stem cell deficiency, with the sandwich approach potentially offering long-term benefits because of the increased presence of progenitor cells in the transplanted cornea.