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Y. Ishino, Y. Sano, T. Horikiri, S. Kinoshita; Cultivated Human Corneal Endothelial Cell Transplantation Using Freeze–Dried Amniotic Membrane as a Carrier . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4716.
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Purpose: We reported cultivated human corneal endothelial cell (cHCEC) transplantation using amniotic membrane (AM) as a carrier and the function of cHCECs in vivo (Y Ishino et al, 2004 IOVS). For cHCEC transplantation, ideally, the carrier should be sterilized and more transparent. Therefore, we wished to examine the feasibility of freeze–dried AM (FD–AM) as a carrier for cHCEC. Methods: cHCECs (10 years old, male) from 6 passages were trypsinized, centrifugated and resuspended at a final cell seeding concentration of 6.0×103cells/mm2. Human AM deprived of amniotic epithelial cells by incubation with EDTA was freeze dried, vacuum packed, and sterilized with gamma–irradiation. On the resultant FD–AM, the cHCEC suspension was seeded, centrifugated, and cultivated for 7days at 37°C, 5%CO2. Cultures of cHCECs on FD–AM were stained with alizarin red and hematoxylin and eosin, and examined by light microscopy. For transplantation experiments in vivo, male Japanese white rabbits were used. cHCEC sheets using FD–AM as a carrier were transplanted onto stroma of excised corneal buttons after stripping Descemet’s membrane. The corneal buttons with cHCEC sheets were then placed on the graft bed of the same animal and sutured. Examination of the corneal appearance via slit–lamp biomicroscopy and ultrasonic pachymetery was procedured, daily up to Day 7 after the operation. Results: The density of cHCECs seeded on FD–AM was 2340.4± 118.3 cells/mm2, fairly continuous monolayer of flat, squamous, polygonal cHCECs appeared on FD–AM. In in vivo study, control grafts consisting of transplanted FD–AM only, after stripping Descemet’s membrane became highly edematous (Corneal thickness; Day 7 after operation: 820.5± 62.9µm), neverthless grafts with cHCECs on FD–AM had little edema and excellent transparency (Corneal thickness Day 7 after operation: 374.7± 53.2µm). Conclusions: We made cHCEC sheets using FD–AM as a nonsynthetic carrier. The morphology and structure of cHCECs transplanted on FD–AM were similar to that of HCECs in vivo. In in vivo study, FD–AM retained more transparency compared with AM, and transplanted cHCECs were functional. We think FD–AM has the feasibility of being a carrier of cHCEC transplantation for corneal endothelial diseases.
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