all the procedures conformed to the principles embodied in the arvo statement for the use of animals in ophthalmic and vision research . parallel lamellar dissection along the limbus ( 2 - 3 mm width ) was done using a beaver knife in right eyes following administration of muscular injections of ketamine ( 30 mg / kg ) and xylazine ( 5 mg / kg ) and topical anesthetic ( 0.5% proparacaine hcl ) on 10 white rabbits weighing 2.0 - 3.0 kg ; in all , 10 limbal tissues were obtained from rabbits . the tissues were washed 6 - 8 times with phosphate buffer containing 5% penicillin / streptomycin , and cut into peices ( 1 mm1.5 mm2.5 mm ) . each tissue piece was treated with 1.2u / ml of dispase ii at 37 or 4 for 1 h or 16 h , respectively . subsequently , the collected sheets of epithelium were treated with 0.25% trypsin/0.01% edta ( gibco . usa ) at 37 for 2 h to generate isolated cells . in another treatment , tissues were treated with 0.05% trypsin and 0.01% ethyldiaminetetraacetic acid ( edta ) at 37 with gentle agitation ( hb - movcst 220 , hybaid limited , u.k ) and vortexing ( vortex genie 2 , scientific industries , u.s.a . ) ; suspended cells were collected 4 times every 20 minutes , and the total exposure time for suspended cell was only 20 minutes . following collection , single cells were stained with trypan blue and counted to determine the numbers of viable cells obtained by processing . each extraction method was repeated 5 times , and a statistical analysis was performed on a personal computer using sigmaster ver 1.0 : spss software ( chicago , il , usa ) . a non parametric mann - whitney u test was performed to evaluate the statistical significance of the differences in viable cell numbers obtained by each method , and a value of p<0.05 was considered significant . nih/3t3 ( atcc , usa ) were used as a feeder layer , treated with 4ug / ml mitomycin c ( sigma.usa ) for 37 for 2 hours , and were plated at 80% confluence of an epithelial cell co - culture . suspended cells were seeded at 1.510 cells / cm , and were primarily cultured with shem media ( dmem / f12 2:1 , 10% fbs , 10 ng / ml egf , 5 ug / ml insulin , 0.1 nm choleratoxin , 50 iu / ml penicillin - streptomycin , 0.18 mm adenine , 4 mm glutamine , hydrocortisone 0.4ug / ml , and 2 nm triiodothyronine ) . for the colony forming assay , primarily cultured cells were plated onto a 3t3 feeder layer at either 100 , 300 , or 600 cells / well in 6-well plate for 9 to 14 days . following formation , colonies were fixed with 4% formaldehyde and stained with 1% rhodamin blue to determine colony counts . for facs of corneal limbal epithelial stem cells , primarily cultured cells were isolated with a flow cytometer , washed with phosphate buffer , treated with 0.02% edta solution to remove the feeder cells , and then with 0.25% trypsin / edta to harvest the cells . suspended cells ( 110 cells / ml ) were treated with 5ug / ml bisbenzimide h 33342 ( hoechst 33342:sigma ) at 37 for 30 min and re - washed with shem media and incubated for a 45-minute efflux period . non - viable cells were excluded by staining with 5 ug / ml propidium iodide ( sigma ) at 37 for 1 min . viable cells were washed twice with shem media and suspended in 3 ml medium for flow cytometry ( coulter epics 753 ) . hoechst 33342 and propidium iodide ( pi ) were stimulated with uv 100 mw ( 351 - 364 nm ) . for hoechst negative cells , hoechst fluorescence was measured using a 424/44 nm bp filter , and to sort the hoechst negative fraction as putative limbal epitheal stem cells ( plescs ) , pi was estimated using a 630/22 nm bp filter . the forward scatter was proportional to cell size , and side scattering was proportional to intracellular dye granularity of hoechst blue . for gating of sp cells , the suspended cells ( 110 cells / ml ) were incubated for a 45-minute efflux period after hoechst treatment . verapamil ( 50 um , sigma ) was used to evaluate the sp in which efflux activity was inhibited . to evaluate the sp , a band pass ( bp ) filter of 424/44 nm and long pass filter of 675 nm were used in combination with a 640 nm long pass dichroic mirror . pi was read concomitantly using a 630/22 nm bp filter and a 610 nm short pass dichroic mirror . forward and side scatter was proportional to the intracellular dye granularity of hoechst red and blue , respectively . the cells in each fraction were seeded onto 12-well plates ( hoechst negative fraction ) and 6-well plates ( hoechst positive fraction ) , with a mitomycin c - treated nih/3t3 ( atcc ) feeder layer , and were primarily cultured with shem media . colony forming efficiencies were evaluated as described above in each fraction sorted by hoechst exclusion . in accordance with the tenets of the declaration of helsinki and with proper informed consent , 10 human corneoscleral rims at 5 days post - harvest were obtained from the northwest lions eye bank within 8 hours after penetrating keratoplasty . all the procedures conformed to the principles embodied in the arvo statement for the use of animals in ophthalmic and vision research . parallel lamellar dissection along the limbus ( 2 - 3 mm width ) was done using a beaver knife in right eyes following administration of muscular injections of ketamine ( 30 mg / kg ) and xylazine ( 5 mg / kg ) and topical anesthetic ( 0.5% proparacaine hcl ) on 10 white rabbits weighing 2.0 - 3.0 kg ; in all , 10 limbal tissues were obtained from rabbits . the tissues were washed 6 - 8 times with phosphate buffer containing 5% penicillin / streptomycin , and cut into peices ( 1 mm1.5 mm2.5 mm ) . each tissue piece was treated with 1.2u / ml of dispase ii at 37 or 4 for 1 h or 16 h , respectively . subsequently , the collected sheets of epithelium were treated with 0.25% trypsin/0.01% edta ( gibco . usa ) at 37 for 2 h to generate isolated cells . in another treatment , tissues were treated with 0.05% trypsin and 0.01% ethyldiaminetetraacetic acid ( edta ) at 37 with gentle agitation ( hb - movcst 220 , hybaid limited , u.k ) and vortexing ( vortex genie 2 , scientific industries , u.s.a . ) ; suspended cells were collected 4 times every 20 minutes , and the total exposure time for suspended cell was only 20 minutes . following collection , single cells were stained with trypan blue and counted to determine the numbers of viable cells obtained by processing . each extraction method was repeated 5 times , and a statistical analysis was performed on a personal computer using sigmaster ver 1.0 : spss software ( chicago , il , usa ) . a non parametric mann - whitney u test was performed to evaluate the statistical significance of the differences in viable cell numbers obtained by each method , and a value of p<0.05 was considered significant . nih/3t3 ( atcc , usa ) were used as a feeder layer , treated with 4ug / ml mitomycin c ( sigma.usa ) for 37 for 2 hours , and were plated at 80% confluence of an epithelial cell co - culture . suspended cells were seeded at 1.510 cells / cm , and were primarily cultured with shem media ( dmem / f12 2:1 , 10% fbs , 10 ng / ml egf , 5 ug / ml insulin , 0.1 nm choleratoxin , 50 iu / ml penicillin - streptomycin , 0.18 mm adenine , 4 mm glutamine , hydrocortisone 0.4ug / ml , and 2 nm triiodothyronine ) . for the colony forming assay , primarily cultured cells were plated onto a 3t3 feeder layer at either 100 , 300 , or 600 cells / well in 6-well plate for 9 to 14 days . following formation , colonies were fixed with 4% formaldehyde and stained with 1% rhodamin blue to determine colony counts . for facs of corneal limbal epithelial stem cells , primarily cultured cells were isolated with a flow cytometer , washed with phosphate buffer , treated with 0.02% edta solution to remove the feeder cells , and then with 0.25% trypsin / edta to harvest the cells . suspended cells ( 110 cells / ml ) were treated with 5ug / ml bisbenzimide h 33342 ( hoechst 33342:sigma ) at 37 for 30 min and re - washed with shem media and incubated for a 45-minute efflux period . non - viable cells were excluded by staining with 5 ug / ml propidium iodide ( sigma ) at 37 for 1 min . viable cells were washed twice with shem media and suspended in 3 ml medium for flow cytometry ( coulter epics 753 ) . hoechst 33342 and propidium iodide ( pi ) were stimulated with uv 100 mw ( 351 - 364 nm ) . for hoechst negative cells , hoechst fluorescence was measured using a 424/44 nm bp filter , and to sort the hoechst negative fraction as putative limbal epitheal stem cells ( plescs ) , pi was estimated using a 630/22 nm bp filter . the forward scatter was proportional to cell size , and side scattering was proportional to intracellular dye granularity of hoechst blue . for gating of sp cells , the suspended cells ( 110 cells / ml ) were incubated for a 45-minute efflux period after hoechst treatment . verapamil ( 50 um , sigma ) was used to evaluate the sp in which efflux activity was inhibited . to evaluate the sp , a band pass ( bp ) filter of 424/44 nm and long pass filter of 675 nm were used in combination with a 640 nm long pass dichroic mirror . pi was read concomitantly using a 630/22 nm bp filter and a 610 nm short pass dichroic mirror . forward and side scatter was proportional to the intracellular dye granularity of hoechst red and blue , respectively . the cells in each fraction were seeded onto 12-well plates ( hoechst negative fraction ) and 6-well plates ( hoechst positive fraction ) , with a mitomycin c - treated nih/3t3 ( atcc ) feeder layer , and were primarily cultured with shem media . colony forming efficiencies were evaluated as described above in each fraction sorted by hoechst exclusion . the number of epithelial cells collected after treatment of limbal tissue with dispase ii and incubation at 37 for 1 h or 4 for 16 h was 1.151.2010 cell / ml and 5.934.010 cell / ml , respectively . the latter method showed better efficacy than the former , with a statistically significant difference ( p<0.028 , mann - whitney u test ) . the number of the cells obtained with a trypsin / edta only treatment was 1.280.5410 cell / ml , which was larger than the number isolated with dispase treatment at 4 for 16 h ; this difference was statistically significant ( p<0.011 , mann - whitney u test , fig . the limbal epithelial cells in rabbits or humans were primarily cultured on plates with mitomycin c - treated nih/3t3 feeder layers . the primarily cultured cells formed abundant colonies with well maintained round shapes , exhibiting the characteristics of decreased cytoplasm and smaller size consistent with stem cells ( fig . the hoechst 33342 exclusion assay revealed that hoechst negative cells with low hoechst blue and red were 3.610.42% and 5.214.91% in rabbit and human , respectively ( fig . each fraction of rabbit cells isolated by hoechst exclusion was cultured until p4 ( nearly 28 days ) . the hoechst negative fraction maintained the small and round morphology well , and formed colonies at high cfes . however , while the hoechst positive fraction formed colonies , the cells became large in size and were differentiated in colonies with low cfes ( fig . the cfes of hoechst negative and hoechcst positive fraction after facs in rabbit were 12.672.24% and 1.176.13% , respectively ( p<0.05 , mann whitney u test ) . the hoechst positive cell fraction grew relatively rapidly and was over 85% of confluence between 6 and 7 days of culture , while the hoechst negative cell fraction grew very slowly and displayed a similar confluence between about 9 and 12 days of culture . our results suggest that corneal epithelial stem cells can be efficiently isolated by an optimal extraction method and facs analysis . in the sampling process of cells from the tissue , it was very important to cause only minimal damage to basal cells , located in the most basal stratum , where epithelial stem cells exist . following the verification of the usefulness of dispase by gipson and grill,23 many researchers have isolated epithelial cells from the cornea by treating the cells with dispase ( 1.2 iu / ml ) at 37 for 1 h.24 - 26 however , metabolism enzymes , which are be easily activated at this temperature , may damage the sensitive stem cells . therefore , we attempted to isolate epithelial cells at 4 , expecting an inhibition of metabolism enzyme activity . indeed , we could isolate nearly 5.2 times more limbal epithelial cells from human tissues with this method . espana et al27 report that the high viability of human limbal epithelial cells may be achieved at culture condition of 4 , which is consistent with our results . to isolate single cells from the sheets obtained using dispase , additional treatment with trypsin - edta was necessary . however , this double procedure by application of dispase and trypsin - edta may have caused more damage to stem cells , even in low temperatures , than would have been caused by a single step procedure . to avoid this possible damage , we simplified the extraction method by using only trypsin - edta ; this single isolation method was used in dealing with epithelial stem cells.28 in this method , continuous agitation and intermittent vortexing was used to isolate single cells , and suspended cells were collected at every 20 minutes to avoid further exposure to trypsin - edta . the yield was 2.2 times higher with a single procedure using trypsin - edta at 37 when compared to the double procedure using dispase at 4 , 16hours and trypsin - edta at 37. facs with hoechst 33342 has been attempted to separate the stem cells of neural29 and dermal20 tissues . these stem cells have been reported to hold an active pump14 ( i.e. abcg2 transporter ) to rapidly release toxic substances in order to maintain stem - cell properties . the abcg2 transporter , which effluxes hoechst dye , is preferentially expressed in stem cells , and sharply down - regulated after commitment to lineage - specific development.14 actually , sp cells or stem cells isolated from the epidermal skin cells of a rat with facs using hoechst 33342 is 3.4% , containing over 95% of label - retaining cells , which allows for positive identification of sp cells as stem cells.20 recently , sp fractions were also found in corneal epithelial cells,21,22,30 however , the gated yield was usually reported as less than 1% ; this finding is consistent with our results . therefore , we adopted hoechst exclusion based only on ability of stem cells to exclude hoechst , and investigated cfe in gated cells after hoechst exclusion to validate proliferation and maintenance capacities of the stem cells . in this study , the cfes of hoechst negative and positive fraction after facs in rabbit were 12.672.24% and 1.176.13% , respectively ( p<0.05 ) . these results suggest that the method can successfully sort cells with higher colony forming abilities . cfe was 9.672.13% in rabbit before facs . likewise , hoechst exclusion efficiently gated putative epithelial stem cells for further use in tissue engineering applications . hoechst negative fractions also developed slow growing small and round shaped colonies in subcultures , suggesting that these cells may also have stem cell properties . however , the hoechst positive cells formed rapidly growing colonies with a much higher level of differentiation . likewise , the sizes of colonies were smaller in sorted cells than those in primary cultured cells , suggesting the possibility of some kind of damage from either uv exposure , long journey time in suspension , and possible hoechst toxicity caused by the sorting procedure . in fact , machalinski b et al report that stem cells damage might possibly be induced by the toxicity of hoechst 33342 itself24 or by the uv detector.31 another problem regarding evaluation of cfes after facs is a result of the small number of cells in hoechst negative fractions . specifically , we could not evaluate cfes in sp cells because of low cell quantities however , as far as our methods were concerned with the feasibility of hoechst exclusion , the data is worthy of notice despite the fact that we could not directly compare the cfes of the cells sorted by hoechst exclusion with those of sp cells . in fact , our cfes in rabbit cells sorted by hoechst exclusion were higher compared with those in sp cells of rabbits reported by budak et al . ( 2.91.2%)30 our results also provide evidence that putative stem cells have a higher colony forming efficiencies in gated cells with hoechst exclusion . further investigation to confirm the identities of the stem cells isolated with hoechst exclusion the efficacy of isolation was highest with a single extraction method using trypsin / edta to obtain a single cell suspension of limbal epithelial cells . hoeschst - excluding cells showed high cfes maintaining stem cell - like morphologies , suggesting that the single method for the isolation of putative stem cells is feasible . additionally , our results may also assist future studies that examine the characteristics of corneal epithelial stem cells and may be critical to the development of successful corneal epithelial stem cell transplantation methods .
purposeto investigate methods of isolating putative corneal epithelial stem cells from cultured limbal tissue.methodsthree extraction techniques were compared to identify an efficient method of obtaining a large number of viable corneal epithelial stem cells from the limbus . 
 limbal tissues were extracted by incubation at 37 or 4 for 1 or 16 hours , respectively , with 1.2u / ml dispase / trypsin or by treatment with 0.05% trypsin and 0.01% ethyldiaminetetraacetic acid ( edta ) at 37 in single procedure . 
 collected cells were cultured on nih/3t3-seeded plates , and colony forming efficiency ( cfe ) was evaluated . 
 fluorescence activated cell sorting ( facs ) was performed with a coulter epics 753 after incubation with hoechst 33342 and propidium iodide ( pi ) . 
 hoechst negative cells were obtained using gates exhibiting low hoechst blue with a 424/44 nm bp filter . 
 gated cells of each fraction were re - cultured to assess the capability of colony formation.resultsthe mean numbers of viable cells obtained from treatment with dispase and trypsin was 3104 cell / ml and 8.06105 cell / ml at 37and 4 incubations ; the number increased to 1.21106 cell / ml with a trypsin / edta treatment ( p<0.05 ) . 
 cfe was 9.672.13% and 6.632.35% in rabbit and human cells , respectively . 
 likewise , the hoechst negative fraction was 3.610.42% and 5.214.91% in rabbit and human cells , respectively . 
 the sorted hoechst negative cells were cultured through four passages , forming small round colonies . in rabbit cells , the cfes of hoechst negative and positive fractions after facs , were 12.672.24% and 1.176.13% , respectively ( p<0.05).conclusionsputative corneal epithelial stem cells were efficiently isolated from limbal tissue using a trypsin / edta extraction and facs . 
 this technique may be very useful in tissue engineered stem cell therapy .