title: A novel method of isolation, preservation, and expansion of human corneal endothelial cells.
abstract: OBJECTIVE
  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.
authors.affiliation: Ocular Surface Center, Miami, Florida 33173, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.forename: Alfonso L
authors.initials: AL
authors.lastname: Sabater
authors.forename: Ying-Ting
authors.initials: YT
authors.lastname: Chen
authors.forename: Yasutaka
authors.initials: Y
authors.lastname: Hayashida
authors.forename: Szu-Yu
authors.initials: SY
authors.lastname: Chen
authors.forename: Hua
authors.initials: H
authors.lastname: He
authors.forename: Scheffer C G
authors.initials: SC
authors.lastname: Tseng
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D018929:Cell Culture Techniques / Q000379:methods*; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D024141:Collagen Type IV; D017364:Collagenases / Q000494:pharmacology; D018031:Connexin 43; D004728:Endothelium, Corneal / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D019812:Heparan Sulfate Proteoglycans / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D020287:In Situ Nick-End Labeling; D007797:Laminin / Q000378:metabolism; D008565:Membrane Proteins; D008875:Middle Aged; D010750:Phosphoproteins; D014019:Tissue Donors; D014021:Tissue Preservation / Q000379:methods*; D062826:Zonula Occludens-1 Protein
chemical_list: D024141:Collagen Type IV; D018031:Connexin 43; C500216:GJA1 protein, human; D019812:Heparan Sulfate Proteoglycans; D007797:Laminin; D008565:Membrane Proteins; D010750:Phosphoproteins; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; C097043:laminin alpha5; C071927:perlecan; D017364:Collagenases