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 g / 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 l / 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 g / ml g418 ( duchefa , amsterdam , netherlands ) was added after 72 hr . one of the established cell lines was seeded onto 100 mm - diameter culture dishes at a density of 510 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 . 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 110 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 g of the total rna in a 20 l 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 m ) . 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 . 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 g / 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 l / 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 g / ml g418 ( duchefa , amsterdam , netherlands ) was added after 72 hr . one of the established cell lines was seeded onto 100 mm - diameter culture dishes at a density of 510 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 . 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 110 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 g of the total rna in a 20 l 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 m ) . 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 . 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 . 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 . 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 , 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 . 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 . 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 . 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 . although the condition of the corneal endothelium is essential for corneal transparency , it is difficult to treat endothelial - damging corneal diseases , since the corneal cells have a very restricted regenerative capacity . in order to overcome these limitations , corneal endothelial cell transplantation had been attempted by several groups , but insurance of a sufficient number of endothelial cells and the development of appropriate endothelial substrates are still not guaranteed . therefore , this experiment was conducted to establish immortalized human corneal endothelial cells ( ihcen ) by stable introduction of hpv 16 e6/e7 , and to investigate the biological characteristics of an established corneal endothelial cell line cultivated on lyophilized human amniotic membrane , an ideal coreneal endothelial substrate . ihcen stably transfected by pa317 amphotropic packaging cell lines with plxsn 16 e6/e7 showed stable expression of e6/e7 mrna . there was no corneal epithelial cell contamination observed by rt - pcr analysis of keratin 12 , a corneal epithelial cell marker ( fig . 2 ) . ihcen showed morphological similarity with phcen , a longer life span ( over passage 30 ) , and more rapid proliferation than phcen ( data not shown ) . 3 ) , was faster than that of a previously established rabbit corneal endothelial cell line in our laboratory ( 51.307.3 hrs).26 several immortalization techniques on human corneal endothelial cells by transfection of various oncogenes including sv40 large t - antigen or hpv 16 e6/e7 had been previously reported,27,28 but there were no reports elucidating the typical cell growth properties such as growth curve or doubling time . introduction of e6/e7 oncogenes facilitates the degradation of p53 and progression into the s phase synergistically.29 - 32 moreover , their stability was also confirmed by observing which gene was non - carcinogenic when injected into nude mice.33 therefore , we were able to successfully obtain abundant populations of highly proliferative and stably immortalized human corneal endothelial cells . furthermore , it was also identified that established ihcen maintained normal corneal endothelial functions similar to phcen by confirming vdac3 , clcn3 , clc4a4 , and na / k atpase mrna expressions ( fig . corneal transparency is maintained by uniform structure , avascularity , and hydration of the stroma . the mechanism that maintains corneal hydration has an active component , hco3- , which is transported from the stroma to the aqueous humor . a number of different hypotheses have been proposed to describe the molecular mechanisms that drive the net hco3 flux . common to these models is the recognition that the net hco3 flux is coupled to and energized by a basolateral na / k atpase in the corneal endothelium.34 the tendency to swell is due to the presence in the stromal matrix of non - diffusible , negatively charged molecules such as glycosaminoglycans . ion transport across the endothelium is thought to involve the active transport of anions from the stroma towards the aqueous humor , followed by the mainly passive diffusion of cations . the osmotic effect of this transendothelial ion transport counters the excess osmotic potential of the stroma.34 at hydration levels outside the normal range , the cornea loses its transparency . thus the positive expressions of several channel proteins including vdac3 and na / k atpase mrna in ihcen means that stably established ihcen did not lose their normal endothelial functions , and can maintain them during ex vivo expansion . essential factors for normal endothelial functions such as channel proteins or na / k atpase were positively expressed in cultivation not only on plastic culture dishes , but also on lam . furthermore , the expression patterns on lam were stronger than that on plastic culture dishes . these results suggest that human amniotic membrane can act as the ideal endothelial niche for ihcen and phcen . the amniotic membrane has a specific advantage that makes it a good substrate for endothelial cell growth . it is a biomaterial different from biochemical substrates such as gelatin membrane or coated hydrolens , it increases cell adherence by abundant extracellular matrix proteins including integrin and lamin , and is easily transplanted since its physical properties are similar to descement 's membrane . furthermore , our results demonstrate that amniotic membrane acts as a good substrate for the maintenance of corneal endothelial functions . this study indicates that a sufficient immortalized cell line having characteristics similar to phcen is useful for in vitro studies of human corneal endothelial functions . furthermore , it is expected that the culture of corneal endothelial cells on an amniotic membrane , an ideal corneal endothelial niche , is useful for the basic investigation of not only drug response or toxicological evaluation , but also for corneal endothelial cell transplantation or the development of reconstructed bioartificial cornea .