Optimizing the role of limbal explant size and source in determining the outcomes of limbal transplantation: An in vitro study

PURPOSE: Simple limbal epithelial transplantation (SLET) and cultivated limbal epithelial transplantation (CLET) are proven clinical techniques for treating limbal stem cell deficiency (LSCD). However, the ideal size and number of the limbal explants required for transplantation has not been clearly elucidated. This in vitro study aimed to determine the optimal limbal explant size required for complete corneal epithelialization by characterizing the cell expansion. METHODS: Limbal explants obtained from both live and cadaveric biopsies were cultured on the denuded amniotic membrane. Explant size and the explant cell outgrowth (expansion) were measured using ImageJ software with respect to days. Cultures were characterized by assessing the rate of proliferation of cells with 5-bromo-2’-deoxyuridine (BrdU) assay along with the expression of different stem cell markers (ABCG2, p63α), corneal epithelial (CK3+12) and adherens junction molecules (E-Cadherin) by immunofluorescence. RESULTS: Explants from live biopsies had 80% growth potential in vitro whereas 40% of the cadaveric tissue failed to grow. Minimum explant sizes of 0.3 mm(2) for live and ≥0.5 mm(2) for cadaveric tissue had a mean expansion areas of 182.39±17.06 mm(2) and 217.59±16.91 mm(2) respectively suggesting adequate growth potential of the explants. Mean total percentage of proliferative cells was 31.80±3.81 in live and 33.49±4.25 in cadaveric tissue expansion. The expression was noted to be similar in cells cultured from cadaveric compared to cells cultured from live limbal tissue with respect to ABCG2, p63α, CK(3+12) and E-cadherin. CONCLUSION: Our findings show that a minimal amount of 0.3 mm(2) live tissue would be sufficient for ample limbal cell expansion in vitro. Cadaveric explants <0.5 mm(2) had poor growth potential. However, larger explants (≥ 0.5 mm(2)) had growth rate and proliferative potential similar to the live tissue. These findings could prove to be critical for clinical success especially while attempting cadaveric limbal transplantation. This study provides a novel clinical strategy for enhancing efficacy of the limbal transplantation surgery and opens the probability of even using the cadaveric tissue by considering the size of explant.