Comparison of Explant and Enzyme Digestion Methods for Ex Vivo Isolation of Limbal Epithelial Progenitor Cells

ABSTRACT

Purpose: To compare the effectiveness of two isolation systems for stemness, proliferation and mesenchymal contamination of ex vivo cultured limbal epithelial cells (LECs).

Methods: Whole explant and dispase digestion methods were used to isolate LECs. For whole explant isolation, one limbal explant was cultivated up to four consecutive times (through LEC1 to LEC4). The performance of LECs isolated with both systems was evaluated according to the following parameters: immunofluorescent staining for adenosine 5′-triphosphate-binding cassette member 2 (ABCG2), p63, cytokeratin 3 (K3), Ki67, and vimentin, and flow cytometry analysis for ABCG2, Ki67 and vimentin.

Results: Twelve LEC cultures were established using whole explant isolation, and nine LEC cultures were established using dispase digestion isolation. In immunofluorescent staining analysis, the ABCG2, p63 and Ki67 expressions were higher in LECs isolated with dispase compared to any LECs isolated with explant. Only the differences in ABCG2 and Ki67 were statistically significant. Further, LEC4 isolated with explant had the highest percentage of cells positive for vimentin, and LEC1 had the highest percentage of cells positive for K3. However, no significant differences were detected. In flow cytometry analysis, the expressions of ABCG2 and Ki67 were statistically higher for LECs isolated with dispase compared to any LECs isolated with explant.

Conclusion: Dispase digestion isolation technique was significantly superior to explant isolation techniques in terms of progenitor and proliferative cell contents.

• Dispase II
• enzyme digestion
• explant
• limbal epithelial cells
• flow cytometry

DECLARATION OF INTEREST

No authors have financial/conflicting interests to disclose. There was no funding or financial support.