a tripartite fiv vector system was used to produce vesicular stomatitis g ( vsv - g ) pseudotyped lentiviral vectors consisting of envelope plasmid pmd.g , packaging plasmid pfp93 , and fiv transfer vector ginsin - encoding enhanced green fluorescent protein ( egfp ) and neomycin resistance via an internal ribosomal entry site ( ires ) .
lentiviral particles were generated using a new , scaled up transient transfection system with polyethylenimine ( pei ) as a polycationic , polymeric transfection agent to provide a simplified and stable platform that allows three full harvests from a single transfection .
prior to transfection , 2.5  10 293 t cells that were cycling at a high cell division rate were seeded in a large surface culture vessel ( cell factory 10 ; nunc , naperville , il , usa ) with 2 l of dulbecco 's modified eagle 's medium ( dmem with 4.5% glucose , 4 mm l - glutamine , without phenol red , without sodium pyruvate ; # sh30284.02 , hyclone ; ge healthcare life sciences , piscataway , nj , usa ) at 37c and supplemented with 10% fetal calf serum and penicillin g sodium / streptomycin sulfate ( 100 units / ml and 100 g / ml , pen strep , life technologies , grand island , ny , usa ) . on the day of transfection ( day 0 )
this transfection mix contained 116 g of vsv - g envelope plasmid pmd - g , 339 micrograms of packaging plasmid pfp93 , and 339 micrograms of transfer plasmid ginsin which were suspended in 18 ml of 37c reduced , serum - free media ( opti - mem ; gibco , thermo fisher scientific , waltham , ma , usa ) that had been combined with 3.2 ml of pei ( polyethylenimine , linear ; polysciences , inc .
, warrington , pa , usa ) , had been vortexed for 10 seconds , and then allowed to stand for 5 minutes at room temperature .
this solution was then pipetted into 1.4 l of fresh media which replaced the one present in the cell factory by gently pouring it along the exchange port without disturbing the cell layers .
the cell factory was balanced to distribute the media equally between the layers and placed in an incubator . following an 8-hour incubation at 37c ,
expression of egfp was imaged by fluorescence microscopy to confirm a transfection rate above 80% . on days 2 , 4 , and 6 ,
vector particles were pelleted by ultracentrifugation in a large volume rotor ( type 19 ; beckman coulter life sciences , brea , ca , usa ) , as previously described , and titrated 24 hours later by fluorescence - assisted flow cytometry ( facs ) analysis on crfk cells . porcine eyes were obtained from the local abattoir and processed within approximately 2 hours of death .
the eyelids and adnexal structures were excised , eyes dipped into 5% povidone - iodine ophthalmic solution ( betadine 5% ; alcon , fort worth , tx , usa ) for 30 seconds and then transferred into sterile pbs ( dulbecco 's pbs ; mp biomedicals , llc , santa ana , ca , usa ) . in a tissue culture hood , the eyes were hemisected along the equator followed by removal of the vitreous , lens , ciliary body , iris , retina , and choroid .
pigment shedding was avoided by carefully dissecting the choroid off the sclera directly along the equatorial incision in one piece while leaving the vitreous and lens in place as a barrier toward the anterior chamber .
the anterior segments were then immediately mounted in perfusion chambers connected to a microinfusion pump ( phd 22/2000 ; harvard apparatus , holliston , ma , usa ) and perfused with serum - free media without phenol red ( dmem with penicillin g sodium / streptomycin sulfate [ 100 units / ml and 100 g / ml , respectively ] ) at a constant flow rate of 3 l / min .
the intraocular pressure was continuously monitored with pressure transducers ( physiological pressure transducer , sp844 ; memscap , skoppum , norway ) and recorded using a software system ( labchart ; adinstruments , colorado springs , co , usa ) .
the perfusion system was calibrated using a pressure transducer tester ( veri - cal ; utah medical products , midvale , ut , usa ) .
eyes that experienced a contamination , showed erroneous iop recordings in the negative pressure range , or readings above 30 mm hg during the first 24 hours were considered a failure .
such negative iop recordings were observed for instance when debris blocked the transducer lumen while early high iop was interpreted as relative tm failure or blockage .
gravity perfused anterior segments ( cogr ) were similarly processed and mounted in perfusion chambers connected to a gravity flow system and perfused with serum free clear dmem supplemented with penicillin g sodium and streptomycin sulfate ( 100 units / ml and 100 g / ml , respectively ) .
the gravity flow system utilized a fluid column at a constant height of 20.4 cm above perfusion chambers to maintain pressures at 15 mm hg .
porcine anterior segments were allowed to stabilize at a constant flow rate for 48 hours before any manipulation . on day 0 , the central infusion and transducer lines were disconnected and a bolus of 1 ml of dmem with 1  10 transducing units ( tu ) ginsin was injected at a rate of 1ml / min , displacing the same amount of intracameral volume .
we redesigned established anterior chamber perfusion dishes by reducing the thickness of the bottom to approximately one - third and widening the now flatter central mounting hub ( fig .
there was reduced image distortion compared with regular chambers with a thick bottom . on days 1 , 3 , 7 , and 14 ,
anterior segments mounted as described above were inverted and placed under a stereo dissecting microscope equipped for fluorescent imaging ( olympus szx16 with gfp filter cube and dp80 monochrome / color camera ; olympus corp . , center valley , pa , usa ) with continued perfusion .
the dish was stabilized at a 30 angle and each quadrant was imaged using an exposure of 100 ms , 1360  1024 resolution and 8-bit depth .
( b ) view through bottom of inverted dish allows the observation of the tm directly with central in - line and peripheral pressure transducer line .
fluorescence of tm was graded on a scale of 0 to 4 as previously established : grade 0 , no detectable fluorescence ; grade 1 , single fluorescent spots in the tm ; grade 2 , numerous , nonconfluent fluorescent spots with some confluent areas ; grade 3 , extensive , mostly confluent , midlevel transduction ; grade 4 , extensive , high - level , and confluent fluorescence . at the conclusion of the experiment
, the anterior segments were removed from the perfusion dish , rinsed in pbs , cut into quadrants , and fixed with 4% paraformaldehyde in pbs for a period of 48 hours before being placed in 70% ethanol .
paraffin - embedded specimens were cut at 6-m thickness and stained with hematoxylin eosin ( h&e ) .
the ratio of transduced to total tm cells was determined as described previously by counting all tm cells in each of four sections per eye ( one from each quadrant ) .
the trabecular meshwork was identified and cell counts of 6-m h&e - stained paraffin sections obtained for two adjacent quadrants in four transduced and nontransduced anterior segments .
photographs of fluorescent tm were taken of each quadrant at each examination and grades determined in a masked manner using a previously established continuous grading scale .
total tm cells present in random 6-m sections from above quadrants were determined by counting the labeled and unlabeled cells and calculating the mean value for each quadrant .
an unpaired student 's t - test was used for statistical analysis to compare transduced and nontransduced anterior segments .
because of the linear correlation between transduced cells and grades , expression grades were handled as continuous data during the statistical comparison of marker protein accumulation .
a tripartite fiv vector system was used to produce vesicular stomatitis g ( vsv - g ) pseudotyped lentiviral vectors consisting of envelope plasmid pmd.g , packaging plasmid pfp93 , and fiv transfer vector ginsin - encoding enhanced green fluorescent protein ( egfp ) and neomycin resistance via an internal ribosomal entry site ( ires ) .
lentiviral particles were generated using a new , scaled up transient transfection system with polyethylenimine ( pei ) as a polycationic , polymeric transfection agent to provide a simplified and stable platform that allows three full harvests from a single transfection .
prior to transfection , 2.5  10 293 t cells that were cycling at a high cell division rate were seeded in a large surface culture vessel ( cell factory 10 ; nunc , naperville , il , usa ) with 2 l of dulbecco 's modified eagle 's medium ( dmem with 4.5% glucose , 4 mm l - glutamine , without phenol red , without sodium pyruvate ; # sh30284.02 , hyclone ; ge healthcare life sciences , piscataway , nj , usa ) at 37c and supplemented with 10% fetal calf serum and penicillin g sodium / streptomycin sulfate ( 100 units / ml and 100 g / ml , pen strep , life technologies , grand island , ny , usa ) . on the day of transfection ( day 0 )
this transfection mix contained 116 g of vsv - g envelope plasmid pmd - g , 339 micrograms of packaging plasmid pfp93 , and 339 micrograms of transfer plasmid ginsin which were suspended in 18 ml of 37c reduced , serum - free media ( opti - mem ; gibco , thermo fisher scientific , waltham , ma , usa ) that had been combined with 3.2 ml of pei ( polyethylenimine , linear ; polysciences , inc .
, warrington , pa , usa ) , had been vortexed for 10 seconds , and then allowed to stand for 5 minutes at room temperature .
this solution was then pipetted into 1.4 l of fresh media which replaced the one present in the cell factory by gently pouring it along the exchange port without disturbing the cell layers .
the cell factory was balanced to distribute the media equally between the layers and placed in an incubator . following an 8-hour incubation at 37c ,
expression of egfp was imaged by fluorescence microscopy to confirm a transfection rate above 80% . on days 2 , 4 , and 6 ,
vector particles were pelleted by ultracentrifugation in a large volume rotor ( type 19 ; beckman coulter life sciences , brea , ca , usa ) , as previously described , and titrated 24 hours later by fluorescence - assisted flow cytometry ( facs ) analysis on crfk cells .
porcine eyes were obtained from the local abattoir and processed within approximately 2 hours of death .
the eyelids and adnexal structures were excised , eyes dipped into 5% povidone - iodine ophthalmic solution ( betadine 5% ; alcon , fort worth , tx , usa ) for 30 seconds and then transferred into sterile pbs ( dulbecco 's pbs ; mp biomedicals , llc , santa ana , ca , usa ) . in a tissue culture hood , the eyes were hemisected along the equator followed by removal of the vitreous , lens , ciliary body , iris , retina , and choroid .
pigment shedding was avoided by carefully dissecting the choroid off the sclera directly along the equatorial incision in one piece while leaving the vitreous and lens in place as a barrier toward the anterior chamber .
the anterior segments were then immediately mounted in perfusion chambers connected to a microinfusion pump ( phd 22/2000 ; harvard apparatus , holliston , ma , usa ) and perfused with serum - free media without phenol red ( dmem with penicillin g sodium / streptomycin sulfate [ 100 units / ml and 100 g / ml , respectively ] ) at a constant flow rate of 3 l / min .
the intraocular pressure was continuously monitored with pressure transducers ( physiological pressure transducer , sp844 ; memscap , skoppum , norway ) and recorded using a software system ( labchart ; adinstruments , colorado springs , co , usa ) .
the perfusion system was calibrated using a pressure transducer tester ( veri - cal ; utah medical products , midvale , ut , usa ) .
eyes that experienced a contamination , showed erroneous iop recordings in the negative pressure range , or readings above 30 mm hg during the first 24 hours were considered a failure .
such negative iop recordings were observed for instance when debris blocked the transducer lumen while early high iop was interpreted as relative tm failure or blockage .
gravity perfused anterior segments ( cogr ) were similarly processed and mounted in perfusion chambers connected to a gravity flow system and perfused with serum free clear dmem supplemented with penicillin g sodium and streptomycin sulfate ( 100 units / ml and 100 g / ml , respectively ) .
the gravity flow system utilized a fluid column at a constant height of 20.4 cm above perfusion chambers to maintain pressures at 15 mm hg .
porcine anterior segments were allowed to stabilize at a constant flow rate for 48 hours before any manipulation . on day 0 , the central infusion and transducer lines were disconnected and a bolus of 1 ml of dmem with 1  10 transducing units ( tu ) ginsin was injected at a rate of 1ml / min , displacing the same amount of intracameral volume .
we redesigned established anterior chamber perfusion dishes by reducing the thickness of the bottom to approximately one - third and widening the now flatter central mounting hub ( fig .
there was reduced image distortion compared with regular chambers with a thick bottom . on days 1 , 3 , 7 , and 14 ,
anterior segments mounted as described above were inverted and placed under a stereo dissecting microscope equipped for fluorescent imaging ( olympus szx16 with gfp filter cube and dp80 monochrome / color camera ; olympus corp . ,
the dish was stabilized at a 30 angle and each quadrant was imaged using an exposure of 100 ms , 1360  1024 resolution and 8-bit depth .
( b ) view through bottom of inverted dish allows the observation of the tm directly with central in - line and peripheral pressure transducer line .
fluorescence of tm was graded on a scale of 0 to 4 as previously established : grade 0 , no detectable fluorescence ; grade 1 , single fluorescent spots in the tm ; grade 2 , numerous , nonconfluent fluorescent spots with some confluent areas ; grade 3 , extensive , mostly confluent , midlevel transduction ; grade 4 , extensive , high - level , and confluent fluorescence .
at the conclusion of the experiment , the anterior segments were removed from the perfusion dish , rinsed in pbs , cut into quadrants , and fixed with 4% paraformaldehyde in pbs for a period of 48 hours before being placed in 70% ethanol .
paraffin - embedded specimens were cut at 6-m thickness and stained with hematoxylin eosin ( h&e ) .
the ratio of transduced to total tm cells was determined as described previously by counting all tm cells in each of four sections per eye ( one from each quadrant ) .
the trabecular meshwork was identified and cell counts of 6-m h&e - stained paraffin sections obtained for two adjacent quadrants in four transduced and nontransduced anterior segments .
photographs of fluorescent tm were taken of each quadrant at each examination and grades determined in a masked manner using a previously established continuous grading scale .
total tm cells present in random 6-m sections from above quadrants were determined by counting the labeled and unlabeled cells and calculating the mean value for each quadrant .
an unpaired student 's t - test was used for statistical analysis to compare transduced and nontransduced anterior segments .
because of the linear correlation between transduced cells and grades , expression grades were handled as continuous data during the statistical comparison of marker protein accumulation .
vector production using the scaled up pei transfection platform yielded 1.1 tu total on first , 7.2 tu on second and 8.0 tu on third harvest with a titer range of approximately 29% at each time point ( n = 3 ) .
2 ) had an iop of 15.8  1.9 mm hg during constant perfusion at a rate of 3 l / min compared with gravity - perfused control anterior segments with an imputed flow rate of 3.7  1.6 l / min at constant gravity perfusion with a pressure of 15 mm hg .
control anterior segments were cultured in the redesigned culture system ( fig . 1 ) and
pilot anterior segments kept for up to 21 days during initial viability testing ( data not shown ) .
trabecular meshwork could be observed directly through the bottom of the inverted dishes ( fig .
1 ) with best detection of egfp fluorescence when held at a 30 angle ( fig .
view was relatively free of distortion for whole eye and quadrant view at up to 10 magnification but became vertically compressed when viewed at higher magnification or when held at a steeper angle ( fig .
could be seen over 360 but with segmental focus and short areas of nontransduction ( fig .
the most notable iop changes occurred when the perfusion system was disconnected for media change ( fig .
4 ) . intraocular pressure graph of perfused anterior segment controls ( n = 22 , left ) .
direct , repeated visualization of whole eye with view of transduced tm through the bottom of the redesigned perfusion system . left : macroscopic view without magnification .
right : fifty - fold magnified view of tm with only minor image distortion ( tm is 700 m wide ) .
intraocular pressure and directly observed , same eye egfp expression in tm of eyes transduced with 10 tu of egfp - expressing fiv vector at time points indicated .
, porcine tm showed low level egfp marker gene expression after transduction at the highest titer tested , 10 tu ( fig .
expression peaked within 48 hours . at this titer , tm transduction was extensive and could be observed throughout the entire periphery .
the culture system enabled us to find precise landmarks during serial observation as in the example of the less transduced , more pigmented tm in the center of the fluorescent band of tm in figure 5 ( fig .
intraocular pressure and directly observed , same eye egfp expression in tm of eyes transduced with 10 tu of egfp - expressing fiv vector at time points indicated .
anterior segments of ginsin transduced with 10 tu experienced a significant ( p = 0.03 ) but transient posttransduction iop increase of 44% at 24 hours that resolved at 48 hours ( fig .
histology obtained at the conclusion of the experiment indicated a well - preserved tm architecture ( fig .
, these anterior segments experienced a small but significant reduction of iop ( p = 0.03 ) before returning to 14.1  2.4 mm hg , which was not significantly different from control anterior segments ( p = 0.2 ) .
expression was initially faint and could only be observed in some anterior segments ( figs . 4 , 5 ) before increasing . at 24 hours ,
relative ginsin expression grading in anterior segments transduced with 10 tu had an average of 0.5  0.7 ( p < 0.01 ) at 24 hours on a scale from 0 to 4 that increased to 2.6  1.1 at 168 hours ( p < 0.01 ) .
anterior segments transduced with 10 tu had a significantly lower expression than those transduced with 10 tu .
it was 0.2  0.4 at 24 hours which increased to 0.7  1.0 ( fig .
peaks for both titers were achieved at the same observation time point . despite weak or absent fluorescence
, the new culture chambers allowed to identify anatomic landmarks in serial observation ( fig .
expression grades of eyes transduced with 10 and 10 tu ( left , n = 8 for each group ; error bars = sd ) and corresponding tm cellularity ( right , n = 8 eyes for each group with two sections per eye counted from adjacent quadrants ; error bars  sd ) .
there are significant supply challenges for human donor eyes in research which is partially due to the increased rate of corneal endothelium and limbal stem cells transplantation . however , a local abattoir can serve as a source of porcine eyes to produce reliable , high quality anterior segment cultures .
compared with human donor eyes that have a wide age range , disease , and variable time from explantation to culture , the consistency of pig eyes resulted in a considerably lower failure rate in our hands .
low costs and close correlation with human anatomy make pig eyes an attractive model for research and training in ophthalmology .
the pig 's similarity in size and anatomy also led to research into xenotransplantation with a focus on kidney , liver , and cornea and , more recently , the outflow tract . here , we describe a method to track transgene expression in an ex vivo pig eye culture system by direct observation that is made possible by redesigned , thinner culture dishes .
the ability to directly observe the tm from the inside will provide a valuable tool to correlate expression of transgenes in the tm with the resulting segmental outflow changes as measured in quantitative canalograms obtained from the outside ( loewen r , et al .
the intraocular pressure of 15.8  1.9 mm hg we observed at the normal aqueous flow rate of 3 to 4 l / min is close to the physiologic iop of 15.2  1.8 mm hg of pigs and has been used in other porcine ex vivo studies .
this intraocular pressure , obtained with a perfusion rate of 3 l / min , matches our imputed outflow of 3.7  1.6 l / min using the reverse approach of infusing these eyes at 15 mm hg in our constant pressure experiments .
in contrast to these studies , we did not observe any prominent corneal swelling that can occur without a physiological transcorneal pressure gradient that allows proper nutrient transport into the stroma and preservation of normal corneal thickness and contour .
the short time from enucleation to culture and limited exposure to povidone - iodine may have further reduced corneal compromise .
problems encountered in prior use of pig eyes include pigment release resulting in trabecular meshwork obstruction , the larger diameter that not all human eye culture systems can accommodate , and the more plexus - like , discontinuous segments of schlemm 's canal . the discontinuity of schlemm 's canal may be an advantage for new outflow research strategies concerned with focal tm modification by limiting effects to the immediate downstream drainage system and providing the opportunity for localized flow measurements .
segmental flow has recently been observed in other species as well . by dissecting the choroid off the sclera directly along the equatorial incision and using the lens as a barrier toward the anterior chamber
copious irrigation alone was not sufficient to dislodge pigment that had already made it into the tm .
we developed a protocol for scaled - up fiv vector production with pei transfection that provided reliable high titers eliminating the ph and titer variability of traditional production systems using calcium chloride .
because transfected cell layers remained adherent , the vector could be harvested three times for an entire week with only moderate decline of titers .
feline immunodeficiency virus , a non - primate lentiviral vector that has the safety advantage of not being based on a human pathogen , can transduce primate , feline , and rodent trabecular meshwork at a high level .
this observation was principally the same in pig eyes , including a limited and temporary effect on outflow function at the highest titers .
different from prior species , the corneal endothelium and sclera that were in proximity to the tm in pig eyes appeared to be transduced more often .
this could be caused by different flow kinetics due to a relatively large anterior chamber with a less selective contact and perfusion of the target tissue .
this vector does not currently deploy receptor or transcriptional targeting strategies that may become necessary in cytoablative models .
the somewhat lower expression levels compared with human and feline eyes may indicate that lentiviral restriction is present but can be overcome at higher titers .
defense mechanisms to curb lateral intrusion of foreign code from retro - elements are known in many species and the most important restriction factors found in human cells are also present in the pig as a genome search for apobec-3f , trim5 , bst2 , samhd1 , and zap demonstrates .
restriction has not presented an obstacle to lentiviral germline transgenesis with hiv and eiav vectors and should not impede future use of these vectors in anterior segments .
relevant to biosafety in tm engineering studies involving xenotransplantation , the pig genome harbors porcine endogenous retroviruses ( perv - a , -b , and -c ) , but these encounter trim and apobec3-mediated restriction in human cells . in conclusion
, we found that pig eyes can be used as a reliable and high quality source for anterior segment perfusion cultures .
the ability to manipulate and directly observe the tm in this culture system , combined with the relative similarity to human eyes , will provide useful tools for new approaches of bioengineering in glaucoma research .
 purposeto establish a consistent and affordable , high quality porcine anterior segment perfusion and transduction model that allows direct visualization of the trabecular meshwork.methodsporcine anterior segments were cultured within 2 hours of death by removing lens and uvea and securing in a specially designed petri dish with a thin bottom to allow direct visualization of the trabecular meshwork with minimal distortion . 
 twenty - two control eyes ( co ) with a constant flow rate were compared to eight gravity perfused eyes ( cogr , 15 mm hg ) . 
 we established gene delivery to the tm using egfp expressing feline immunodeficiency virus ( fiv ) vector ginsin at 108 transducing units ( tu ) per eye ( ginsin_8 , n = 8) and 107 tu ( ginsin_7 , n = 8) . 
 expression was assessed for 14 days before histology was obtained.resultspig eyes were a reliable source for consistent and high quality anterior segment cultures with a low failure rate of 12% . 
 control eyes had an intraocular pressure ( iop ) of 15.8  1.9 mm hg at fixed pump perfusion with 3 l / min compared to gravity perfused cogr with imputed 3.7  1.6 l / min . 
 vector ginsin_8 eyes experienced a transient posttransduction iop increase of 44% that resolved at 48 hours ; this was not observed in ginsin_7 eyes . 
 expression was higher in ginsin_8 than in ginsin_7 eyes . 
 trabecular meshwork architecture was well preserved.conclusionscompared with previously used human donor eyes , this inexpensive porcine anterior segment perfusion model is of sufficient , repeatable high quality to develop strategies of tm bioengineering . 
 trabecular meshwork could be observed directly . despite significant anatomic differences , effects of transduction 
 replicate the main aspects of previously explored human , feline and rodent models . 
Methods
Feline Immunodeficiency Viral (FIV) Vector Production
Porcine Eye Anterior Segment Perfusion Culture
Transduction
Direct Visualization of the Trabecular Meshwork
Histology
Statistical Analysis
Results
Discussion
Supplementary Material