A novel method of isolation, preservation, and expansion of human corneal endothelial cells.
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.
Li
Wei
W
Ocular Surface Center, Miami, Florida 33173, USA.
Sabater
Alfonso L
AL
Chen
Ying-Ting
YT
Hayashida
Yasutaka
Y
Chen
Szu-Yu
SY
He
Hua
H
Tseng
Scheffer C G
SC
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
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