Document Detail

A novel method of isolation, preservation, and expansion of human corneal endothelial cells.
MedLine Citation:
PMID:  17251457     Owner:  NLM     Status:  MEDLINE    
PURPOSE: To explore new strategies for effective isolation, preservation, and expansion of human corneal endothelial cells (HCECs).
METHODS: Human corneal Descemet's membrane and corneal endothelial cells were digested with collagenase A or Dispase II in supplemented hormonal epithelial medium (SHEM) for 1.5 to 16 hours. HCEC aggregates derived from collagenase A digestion were preserved in serum-free medium with low or high calcium for up to 3 weeks. Cryosections of HCEC aggregates were subjected to immunostaining with ZO-1, connexin 43, type IV collagen, laminin-5, and perlecan, and apoptosis was determined by TUNEL or cell-viability assay. For expansion, HCEC aggregates were seeded directly or after brief treatment with trypsin/EDTA in SHEM, with or without additional bovine pituitary extract (BPE), nerve growth factor (NGF), or basic fibroblast growth factor (bFGF). The resultant HCECs were immunostained with ZO-1, connexin 43, and Ki67.
RESULTS: Digestion with collagenase A, but not Dispase, of the stripped Descemet's membrane generated HCEC aggregates, which preserved cell-cell junctions and basement membrane components. High cell viability of HCEC aggregates was preservable in a serum-free, high-calcium, but not low-calcium, medium for at least 3 weeks. Brief treatment of HCEC aggregates with trypsin/EDTA resulted in a higher proliferation rate than without, when cultured in SHEM, and the resultant confluent monolayer of hexagonal cells retained cell-cell junctions. However, additional BPE, NGF, or bFGF did not increase cell proliferation, whereas additional BPE or bFGF disrupted cell-cell junctions.
CONCLUSIONS: Collagenase A digestion successfully harvested aggregates with viable HCECs that were preservable for at least 3 weeks in a serum-free, high-calcium medium and, with brief trypsin/EDTA treatment, expanded in the SHEM into a monolayer with hexagonal cells that exhibited characteristic cell junctions.
Wei Li; Alfonso L Sabater; Ying-Ting Chen; Yasutaka Hayashida; Szu-Yu Chen; Hua He; Scheffer C G Tseng
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Investigative ophthalmology & visual science     Volume:  48     ISSN:  0146-0404     ISO Abbreviation:  Invest. Ophthalmol. Vis. Sci.     Publication Date:  2007 Feb 
Date Detail:
Created Date:  2007-01-25     Completed Date:  2007-03-01     Revised Date:  2014-09-15    
Medline Journal Info:
Nlm Unique ID:  7703701     Medline TA:  Invest Ophthalmol Vis Sci     Country:  United States    
Other Details:
Languages:  eng     Pagination:  614-20     Citation Subset:  IM    
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MeSH Terms
Cell Culture Techniques / methods*
Cell Separation / methods*
Cell Survival
Collagen Type IV
Collagenases / pharmacology
Connexin 43
Endothelium, Corneal / cytology*,  drug effects,  metabolism
Heparan Sulfate Proteoglycans / metabolism
Immunoenzyme Techniques
In Situ Nick-End Labeling
Laminin / metabolism
Membrane Proteins
Middle Aged
Tissue Donors
Tissue Preservation / methods*
Zonula Occludens-1 Protein
Grant Support
R01 EY006819/EY/NEI NIH HHS; R01 EY015735/EY/NEI NIH HHS; R01 EY015735/EY/NEI NIH HHS; R01 EY06819/EY/NEI NIH HHS
Reg. No./Substance:
0/Collagen Type IV; 0/Connexin 43; 0/GJA1 protein, human; 0/Heparan Sulfate Proteoglycans; 0/Laminin; 0/Membrane Proteins; 0/Phosphoproteins; 0/TJP1 protein, human; 0/Zonula Occludens-1 Protein; 0/laminin alpha5; 143972-95-6/perlecan; EC 3.4.24.-/Collagenases

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