Jae-Il Ahn

A comparison of lyophilized amniotic membrane with cryopreserved amniotic membrane for the reconstruction of rabbit corneal epithelium

Many researchers have employed cryopreserved amniotic membrane (CAM) in the treatment of a severely damaged cornea, using corneal epithelial cells cultured on an amniotic membrane (AM). In this study, two Teflon rings were made for culturing the cells on the LAM and CAM, and were then used to support the AM, which is referred to in this paper as an Ahn’s AM supporter. The primary corneal epithelial cells were obtained from the limbus, using an explantation method. The corneal epithelium could be reconstructed by culturing the third-passage corneal epithelial cells on the AM. A lyophilized amniotic membrane (LAM) has a higher rate of graft take, a longer shelf life, is easier to store, and safer, due to gamma irradiation, than a CAM. The corneal epithelium reconstructed on the LAM and CAM, supported by the two-Teflon rings, was similar to normal corneal epithelium. However, the advantages of the LAM over that of the CAM make the former more useful. The reconstruction model of the corneal epithelium, using AM, is considered as a goodin vitro model for transplantation of cornel epithelium into patients with a severely damaged cornea.

Characterization of Immortalized Human Corneal Endothelial Cell Line using HPV 16 E6/E7 on Lyophilized Human Amniotic Membrane

Purpose To establish the immortalized human corneal endothelial cell line (IHCEn) by transducing human papilloma virus (HPV) 16 E6/E7 oncogenes, and to identify their characteristics when cultivated on a lyophilized human amniotic membrane (LAM). Methods Primary human corneal endothelial cells (PHCEn) were infected using a retroviral vector with HPV 16 E6/E7, and transformed cells were clonally selected by G418. Growth properties and characteristics of IHCEn were compared with PHCEn by cell counting and RT-PCR of VDAC3, SLC4A4, CLCN3, FGF-1, Col IV, and Na+/K+ ATPase. IHCEn were cultured on LAM. Messenger RNA expressions of VDAC3, CLCN3, and Na+/K+ ATPase, and protein expressions of Na+/K+ ATPase and Col IV in IHCEn cultivated on LAM were investigated by RT-PCR, immunofluorescence, and immunohistochemical staining, respectively. Results Successful immortalization was confirmed by stable expression of HPV 16 E6/E7 mRNA by RT-PCR, and IHCEn exhibited typical corneal endothelial morphology. Doubling time of IHCEn was 30.15±10.96 hrs. Both IHCEn and PHCEn expressed VDAC3, CLCN3, SLC4A4, FGF-1, Col IV, and Na+/K+ ATPase. IHCEn cultivated on LAM showed stronger expression of VDAC3, CLCN4, and Na+/K+ ATPase mRNA than on plastic culture dish. Immunohistochemical staining and immunofluorescence revealed the positive expression of Na+/K+ ATPase and Col IV. Conclusions IHCEn were successfully established, and LAM is a good substrate for the culture of human corneal endothelial cells.

The Effect of Lyophilization on Graft Acceptance in Experimental Xenotransplantation Using Porcine Cornea

The immunogenicity of lyophilized porcine cornea is unknown. The purpose of this study was to examine the possibility of using lyophilized porcine cornea as a substrate for ocular surface reconstruction. A porcine cornea stromal button was freeze-dried and vacuum-packed. Lyophilized and fresh porcine corneas were examined histologically, and then implanted into intrastromal pockets in live rat corneas. Cytokine concentrations in plasma and protein extracts from the corneal buttons of rats were measured using the fluorokine multianalyte profiling assay, and histologic examination was performed. Immunoreactivity to the alpha-gal epitope was not found in lyophilized porcine corneas, whereas it was found in several keratocytes in fresh porcine corneas. The median survival time of rat corneas receiving lyophilized porcine transplants was 28.0 days, significantly longer than the 14.0-day survival of rat corneas that received fresh porcine transplants (P < 0.05). CD45RO(+) and CD68(+) cells were observed in rejected corneas, and interleukin-2 and interferon-gamma were elevated in rat plasma and corneal tissue. The lyophilized porcine corneal stroma, which is devoid of alpha-gal epitope, is less antigenic, and may be a useful biomaterial for ocular surface reconstruction and corneal collagen supplementation.

Reconstruction of Rabbit Corneal Layer Composed of Corneal Fibroblasts and Corneal Epithelium on the Lyophilized Amniotic Membrane

Many researchers have employed the cryopreserved amniotic membrane (CAM) and corneal epithelial cells in the treatment of a severely damaged burned cornea, with corneal epithelial cells cultured on an amniotic membrane (AM). The lyophilized amniotic membrane (LAM) has a higher graft take and a longer shelf life; it is easier to store and safer because of gamma irradiation. Two Teflon rings (Ahns supporter) were made for culturing the cells on the LAM, and were then used to support the LAM. To reconstruct a corneal layer composed of corneal fibroblasts and epithelium, the corneal fibroblasts were first cultivated on the stromal side of LAM for five days, followed by epithelial cells culture on the epithelial side, by using the air-liquid interface culture. The reconstructed corneal layer composed of corneal fibroblasts and corneal epithelial cells has a much healthier basal layer of corneal epithelium than the reconstructed corneal epithelium, which was got by using only corneal epithelial cells, and resembles the epithelium of normal corneas, without the horny layer. Thus, the reconstruction of the corneal layer by using a LAM is considered to be a good in vitro model, not only for its application in toxicological test kits, but also for transplantation in patients with a severely damaged cornea.