All materials for cell culture including fetal bovine serum (FBS) and Opti-MEM were obtained from Gibco BRL (Grand Island, NY, USA). Other media supplements, such as epidermal growth factor (EGF), ascorbic acid, RPMI 1640 vitamin mixture, insect lipid, and chondroitin sulfate, were purchased from Sigma Chemical (St. Louis, MO, USA). Nerve growth factor (NGF) was obtained from R&D Systems (Minneapolis, MN, USA).

Human eyes were obtained in accordance with the tenets of the Declaration of Helsinki and proper informed consent. Human corneal endothelial cells were isolated by trypsin digestion of the posterior portions of excised cornea. After enzymatic digestion at 37℃ for 10 to 15 min, the endothelium was removed by gentle scraping, and seeded onto a tissue culture dish in Opti-MEM supplemented with 5 ng/ml of EGF, 20 ng/ml of NGF, 20 µg/ml of ascorbic acid, 0.005% insect lipid, 200 mg/l of CaCl₂, 0.02% chondroitin sulfate, 1% RPMI 1640 vitamin mixture, and 8% FBS.

PA317 amphotropic packaging cell lines stably transfected with pLXSN 16 E6-E7 (HPV 16 E6/E7) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were grown to 70-80% confluence, and supernatants were collected for 24 hr and stored in aliquots at -80℃. The primary endothelial cells were infected with 1 ml of virus stock in 3 ml of medium containing Polybrene (Sigma, MO, USA) at 4 µl/ml for 24 hr. The virus was then removed and the medium replaced by Opti-MEM supplemented with 8% FBS and other supplements as described above. Selection media with 200 µg/ml G418 (DUCHEFA, Amsterdam, Netherlands) was added after 72 hr. Cultures were maintained in selective media for two to three weeks. The G418-selected transformed cells were then grown and expanded further.

One of the established cell lines was seeded onto 100 mm-diameter culture dishes at a density of 5×10⁴ cells/dish. Every two days for 14 days, cells were detached by 0.05% trypsin/0.5 mM ethylenediaminetetraacetic acid (EDTA), and counted in a hemocytometer in triplicate. For doubling time determination, the formula Tc=0.3T/log (A/A₀) was used where Tc=doubling time, T=initial time, A=the number of cells at time T of proliferation, and A0=the number of cells at an initial time point (Table 1).

Human amniotic membrane from donated human placenta tested by hepatitis or HIV infection was pretreated with 0.025% trypsin-EDTA to remove amnion cells, followed by lyophilization and sterilization using EO gas. Lyophilized amniotic membranes (LAM) were installed on a Teflon ring, and 1×10⁵ cells/support IHCEn were seeded onto the amnion side of a denuded LAM. Cells were maintained using opti-MEM for six days by changing the growth media every two days.

Corneal endothelial origin of the primary and transformed cells was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). Primary cells and established cell lines were cultured as above, and total RNA was extracted with Trizol reagent (Gibco BRL, NY, USA). The first strand of cDNA was synthesized from two µg of the total RNA in a 20 µl reaction mixture containing Superscript II RNase, H reverse transcriptase, and oligo (dT)18 primer. Target genes (Table 2) were then amplified, PCR products were run on a preparative 2% agarose gel, and the bands were photographed.

An avidin biotin complex technique was selected for staining sectioned specimens (2-4 µm). Paraffin sections were deparaffinized in xylene, rehydrated through decreasing ethanol concentrations and quenched for endogenous peroxidase. Cryostat sections were placed on gelatinized slides, fixed in cold acetone and then rinsed in tris-buffered saline. Nonspecific background was eliminated by incubating tissue sections with non-immuno serum (Histostatin-plus Kits, Reagent A; Zymed Laboratories, CA, USA). Sections were then incubated with monoclonal mouse anti-human collagen IV (Santa Cruze Biotechnology Inc. CA, USA) and anti-human Na⁺/K⁺ ATPase antibody (Santa Cruze Biotechnology Inc. CA, USA) overnight at 4℃, followed by extensive washing in 0.05 M tris-buffered saline (pH 7.6) before the addition of a biotinylated secondary antibody (reagent B). Sections were washed again and incubated for one hour with peroxidase-conjugated streptavidin (reagent C). The presence of peroxidase was revealed by adding a substrate-chromogen (3-amino-9-ethycarbazole) solution (reagent D) for immunohistochemistry, and an FITC conjugated rabbit anti-mouse IgG for immunofluorescence. Sections were counterstained with haematoxylin. All sections were photographed and the entire tissue area was examined.

The morphological characteristics of isolated primary human corneal endothelial cells (PHCEn) and IHCEn were observed using an inverted microspcope. IHCEn were polygonal to slightly elongated in shape, similar to PHCEn, but were siginificantly different from dendritic corneal fibroblasts and small globular-shaped epithelial cells (Fig. 1). Stable expression of E6/E7 mRNA was observed in IHCEn, but not in PHCEn by RT-PCR analysis. Messenger RNA of the corneal epithelial cell marker, keratin 12, was not expressed in PHCEn and IHCEn (Fig. 2).

Proliferative characteristics of IHCEn were elucidated by cell counting every two days for 14 days. The cell growth curve of IHCEn showed typical S-curves: minimal growth for the initial seven days, geometrical growth until day 14, and decreased cell numbers after 14 days. The estimated cell doubling time of IHCEn was 30.15±10.96 hrs (Fig. 3). IHCEn showed a more extended life span (over passage 30), and more rapid proliferation than PHCEn (data not shown). Transformed traits, except for the transducing of HPV 16 E6/E7 oncogenes, were examined by RT-PCR of several channel proteins including voltage-dependent annion channel 3 (CDAC3), sodium bicarbonate cotransporter member 4 (SLC4A4), chloride channel protein 3 (CLCN3), fibroblast growth factor (FGF)-1, type IV collagen (Col IV), and Na⁺/K⁺ ATPase in PHCEn and IHCEn. mRNAs for all of the mentioned proteins were expressed in IHCEn, with similar expression patterns observed in PHCEn.

In order to elucidate the efficiency of LAM for the human corneal endothelial niche, IHCEn were cultured on LAM and harvested for characterization. Messenger RNA expressions of several channel proteins and Na⁺/K⁺ ATPase were observed in established IHCEn cultivated on LAM. Moreover, their expressions were stronger than in cells grown on a plastic culture dish (Fig. 5). Immunohitochemical staining of Col IV and immunofluorescence of Na⁺/K⁺ ATPase in IHCEn cultivated on LAM also showed positive expressions results similar to RT-PCR (Fig. 6).