title: Comparison of Explant and Enzyme Digestion Methods for Ex Vivo Isolation of Limbal Epithelial Progenitor Cells.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  Dispase digestion isolation technique was significantly superior to explant isolation techniques in terms of progenitor and proliferative cell contents.
authors: a Department of Ophthalmology , Shanghai First People's Hospital Affiliated to Shanghai Jiaotong University , Shanghai , China.
authors: Zhi-Hua
authors: ZH
authors: Zhang
authors: a Department of Ophthalmology , Shanghai First People's Hospital Affiliated to Shanghai Jiaotong University , Shanghai , China.
authors: Hai-Yun
authors: HY
authors: Liu
authors: a Department of Ophthalmology , Shanghai First People's Hospital Affiliated to Shanghai Jiaotong University , Shanghai , China.
authors: Kun
authors: K
authors: Liu
authors: a Department of Ophthalmology , Shanghai First People's Hospital Affiliated to Shanghai Jiaotong University , Shanghai , China.
authors: Xun
authors: X
authors: Xu
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2 / Q000378:metabolism; D000328:Adult; D015415:Biomarkers / Q000378:metabolism; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D010450:Endopeptidases / Q000494:pharmacology; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D019394:Ki-67 Antigen / Q000378:metabolism; D016850:Limbus Corneae / Q000166:cytology* / Q000378:metabolism; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000378:metabolism; D009924:Organ Culture Techniques; D059748:Proteolysis; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism; D055815:Young Adult
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D015415:Biomarkers; D019394:Ki-67 Antigen; D009363:Neoplasm Proteins; C115051:TP63 protein, human; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D014746:Vimentin; D010450:Endopeptidases; C023139:dispase
keywords: Dispase II; enzyme digestion; explant; flow cytometry; limbal epithelial cells