3 days to digest expansion
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Regarding the method of expanding isolated HCECs, a significant advance has been made by Senoo et al., 9showing that the use of EDTA to dissociate cell–cell junctions is essential for the stimulation of cellular proliferation by releasing the cell cycle block. There has not been any attempt made to investigate the method of preservation of isolated HCECs. 7 8However, none have systemically compared these different enzymatic methods to determine the yield and the reproducibility, nor have they considered the possibility that the digestion in a certain medium may trigger cell death. Regarding the method of isolating HCECs from the donor cornea, some have used the nonenzymatic method based on EDTA, 5but most have relied on enzymatic digestion using trypsin/EDTA, Dispase, 6or collagenase. No study has been conducted to address the aforementioned three key steps in a comprehensive manner. 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.
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However, additional BPE, NGF, or bFGF did not increase cell proliferation, whereas additional BPE or bFGF disrupted cell–cell junctions.Ĭonclusions. 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. High cell viability of HCEC aggregates was preservable in a serum-free, high-calcium, but not low-calcium, medium for at least 3 weeks. 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. The resultant HCECs were immunostained with ZO-1, connexin 43, and Ki67. 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).
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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. HCEC aggregates derived from collagenase A digestion were preserved in serum-free medium with low or high calcium for up to 3 weeks. 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. To explore new strategies for effective isolation, preservation, and expansion of human corneal endothelial cells (HCECs).