Article
Development of a murine ocular posterior segment explant culture for the study of intravitreous vector delivery Nora Denk, Vikram Misra, Lynne S. Sandmeyer, Bianca B. Bauer, Jaswant Singh, George W. Forsyth, Bruce H. Grahn
Abstract The objective of this study was to develop a murine retinal/choroidal/scleral explant culture system to facilitate the intravitreous delivery of vectors. Posterior segment explants from adult mice of 2 different age groups (4 wk and 15 wk) were cultured in serum-free medium for variable time periods. Tissue viability was assessed by gross morphology, cell survival quantification, activated caspase-3 expression, and immunohistochemistry. To model ocular gene therapy, explants were exposed to varying transducing units of a lentiviral vector expressing the gene for green fluorescent protein for 48 h. Explant retinal cells remained viable for approximately 1 wk, although the ganglion cell layer developed apoptosis between 4 and 7 d. Following vector infusion into the posterior segment cups, viral transduction was noted in multiple retinal layers in both age groups. An age of donor mouse influence was noted and older mice did not transduce as well as younger mice. This explant offers an easily managed posterior segment ocular culture with minimum disturbance of the tissue, and may be useful for investigating methods of enhancing retinal gene therapy under controlled conditions.
Résumé L’objectif de la présente étude était de développer un système murin de culture d’explant de rétine/choroïde/sclérotique afin de faciliter la livraison intra-vitréenne de vecteurs. Des explants de segments postérieurs provenant de souris adultes de deux groupes d’âge différents (4 sem et 15 sem) furent cultivés dans un milieu sans sérum pour des périodes de temps variables. La viabilité tissulaire fut évaluée par morphologie macroscopique, quantification de la survie cellulaire, expression de la caspase-3 activée, et immunohistochimie. Afin d’imiter la thérapie génique oculaire, les explants furent exposés pendant 48 h à des unités de transduction variables d’un vecteur lentiviral exprimant le gène pour la protéine fluorescente verte. Les explants de cellules de la rétine sont demeurés viables pour environ 1 sem, bien que dans la couche de cellules ganglionnaires on nota le développement de l’apoptose entre 4 à 7 j. Suite à l’infusion de vecteur dans le segment postérieur, la transduction virale fut notée dans plusieurs couches rétiniennes des animaux des deux groupes d’âge. Une influence de l’âge de la souris donneuse fut notée et chez les souris plus âgées la transduction ne se faisait pas aussi bien que chez les jeunes souris. Ce modèle d’explant permet la gestion facile de culture de segment oculaire postérieur avec un minimum de dérangement du tissu, et pourrait être utile pour étudier des méthodes visant à augmenter la thérapie génique sous conditions contrôlées. (Traduit par Docteur Serge Messier)
Introduction Retinal explant culture has been used to study retinal development (1–15), pathophysiological processes of neurodegenerative disease (16–19), central nervous system regeneration (8,13,20–23), cell death and neuroprotection (24–26), electrophysiological recording (27,28), cell transplantation (29–34), test therapeutic substances (16,18,35), examine the role of growth factors (36,37), gene therapy (38), and compare the transduction efficiency of adeno-viral vector mediated gene transfer at different ages in normal mice and mice with retinal degeneration (39). Such studies have involved tissue from embryonic, neonatal animals or from young animals, all of which demonstrate a relatively high level of receptiveness to genetic manipulation compared with adult tissue. We are not aware of any
study that directly compared transduction efficiency in murine posterior ocular explants from 2 age groups. Retinal explant cultures in the first half of the 20th century were primarily grown in plasma clots or in a collagen matrix using a roller tube method, also known as the flying coverslip method, and variations of this method are still used today (11,40–42). In the 1950s, Trowell developed the membrane culture in which the tissue is placed on a porous membrane on top of a wire grid and maintained at the air-medium interface (43). Caffe, et al (44) developed a method in which the neural retina is placed with the vitreous surface facing upwards on rafts made of nitrocellulose filters and polyamide gauze grids. The first retina–retinal pigment epithelium (RPE) culture was reported by Tamai et al (45), and a number of other investigators
Department of Small Animal Clinical Sciences (Denk, Sandmeyer, Bauer, Grahn); Veterinary Microbiology (Misra); and Veterinary Biomedical Sciences (Singh, Forsyth), Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan. Address all correspondence to Dr. Bruce H. Grahn; e-mail: [email protected] Received April 1, 2013. Accepted February 25, 2014. 2015;79:31–38
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Figure 1. This illustration depicts the surgical preparation of freshly enucleated mouse eyes that were used for viral transduction.
have published similar studies of neural retina-RPE cultures from rodents (10,44,46), chickens (47,48), frogs (49,50), and pigs (51). In recent years there have been a number of reports on the culture of isolated neural retina that is not attached to the RPE and choroid. A reduction of apoptosis in retina–RPE culture, compared to cultures of isolated retina culture has been reported (52). Others have described an explant model of whole globes punctured with a needle to enable fluid exchange, although tissue viability was not reported (52,53). Despite promising results in retinal explant culture, the viability and behavior of retinal tissue from adult rodents has not been rigorously evaluated. The integrity of the retina and the ability to genetically modify cells that remain intact are important if this model is to be used in gene therapy development. Intra-vitreous delivery of medications and therapies for acquired and developmental ocular diseases has become a common ophthalmological examination and operating room procedure during the last decade. The development of gene therapy is in its embryologic stages and future development will benefit from coordination of cell culture models that allow for the optimization of vitreous delivery. Posterior segment culture models that facilitate expression/concentration/and vector comparison data, allow for alternative delivery systems, and adjunctive medication strategies prior to in vivo animal model trials are paramount. These techniques will not replace the use of in vivo models; however, they may improve efficiency and reduce the numbers of animals utilized in the future. The purpose of this study was to establish cultures of retina-RPEchoroid-sclera explants from mice of different ages, to define their survival in culture, and to compare transduction efficiency.
Materials and methods Animals Adult c57Bl/6 mice, referred to as wildtype, were obtained from the local animal resource facility. All animals were maintained, treated, and sacrificed in compliance with the Canadian Council of Animal Care Guide to the Care and Use of Experimental Animals, and the University of Saskatchewan Animal Care Committee approved the research protocol. Only mice with eyes that were devoid of any ocular abnormalities based on biomicroscopic and indirect ophthalmoscopic examinations were utilized. Sixteen mice were sacrificed at 2 different postnatal ages (4 and 15 wks of age) by intraperitoneal injection of pentobarbital (100 mg/kg BW), the eyes immediately enucleated postmortem and posterior segment explants were prepared.
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Lentiviral vector system The green fluorescent protein (GFP) coding sequences were amplified by polymerase chain reaction (PCR) from the plasmid pcDNA3.1-GFP and the nucleotide sequence was confirmed by DNA sequencing. The PCR product was cloned into pCR 8-TOPO using a cloning kit (TOPO cloning kit; Life Technologies Inc, Burlington, Ontario) and then transferred to pLenti6.3 (Lentivirus Kit; Life Technologies) using another cloning kit (Gateway cloning kit; Life Technologies). A V5 epitope enables immunolabelling of the gene product. Lentiviral vector was produced following the manufacturer’s instructions using modified and optimized ratios of reagents (Invitrogen, Carlsbad, California, USA). The titer of the virus stock was assessed by transduction of HEK 293 and Vero cells in vitro, followed by immunofluorescence and expressed as transducing units (TU). Manipulations of lentiviral vectors were done in accordance with institutional and national biosafety restrictions and a biosafety permit issued by the University of Saskatchewan Biosafety Committee.
Posterior segment explant preparation and culture Immediately following euthanasia both eyes were enucleated and rinsed with ice-cold balanced salt solution (BSS) containing penicillin (100 U/mL) and streptomycin (100 g/mL). All preparation steps were performed at room temperature. With the aid of an operating microscope scleral incisions were made using 6500 Beaver blades (Lab Tician, Oakville, Ontario) approximately 1 to 2 mm posterior to the limbus of each eye (Figure 1). The stab incision was extended circumferentially with micro scissors to separate the anterior segment of each globe, including the lens and the attached vitreous from the remaining posterior segment. The retina — RPE — choroid — scleral explants were placed in cell culture dishes containing 300 mL of prewarmed media with the inner limiting membrane side facing up (Figure 1). Medium consisted of L-glutamine (0.8 mM) containing neuronal growth medium (Neurobasal A; Invitrogen Ltd.), supplemented with 2% B27 (Invitrogen Ltd.), 1% N2 (Invitrogen Ltd.), penicillin (100 IU/mL), and streptomycin (100 g/mL) as previously described (54). Posterior segment explant cultures were maintained in humidified incubators at 37°C and 5% CO2 for periods of 2 to 7 d. Half of the media were changed every following day. Each culture was repeated 4 times with tissues originating from 4 different mice from 2 different age groups (4 wk and 15 wk of age). Four eyes from age-matched mice that were examined and then euthanized, as previously described, were enucleated and fixed in chilled 4% paraformaldehyde (PFA) and these tissues served as controls. Culture of
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Table I. Details of antibodies and fluorescent markers used for immunohistochemistry Source Specificity Cells labeled (isotype) Conjugate Clone Company Primary antibodies Active caspase-3 Apoptotic cells Rabbit NA Polyclonal Abcam, Cederlane, Burlington, Ontario
Working concentration (mg/mL)
2.0
V5 V5-epitope Mouse NA Polyclonal
Invitrogen, Carlsbad, California, USA
5.0
Secondary antibodies and other labels Rabbit IgG (H 1 L)
NA
Goat
AlexaFluor 488
NA
Invitrogen
2.0
Rabbit IgG (H 1 L)
NA
Goat
AlexaFluor 546
NA
Invitrogen
2.0
Mouse IgG (H 1 L)
NA
Donkey
AlexaFluor 633
NA
Abcam, Cederlane
2.0
Mouse IgG (H 1 L)
NA
Goat
AlexaFluor 488
NA
Invitrogen
2.0
NA
Invitrogen
0.5
DAPI NA Ig — immunoglobulin; DAPI: 49,6-diamidino-2-phenylindole; NA — Not available.
2 whole globes incised only with a perilimbal incision but without separation of the anterior segment was done.
Tissue processing Tissues were fixed by replacing the culture media with freshly prepared 4% PFA for 12 h at 4°C. Whole eyes were enucleated from mice after euthanasia and immersion-fixed in 4% PFA for 12 h at 4°C. The explants and whole eyes were then cryoprotected in 30% sucrose for 24 h at 4°C before being embedded in optimal cutting temperature (OCT) compound (Sakura Finetek USA Inc., Torrance, California, USA), and frozen at 220°C. Posterior segment explants were sectioned vertically [20 mm thickness for immunohistochemistry, 6 mm thickness for hematoxylin and eosin (H&E) staining] on a cryostat microtome at 220°C. For immunohistochemistry, serial sections were mounted on poly-l-lysine coated slides and stored at 220°C. Slides were the washed in 0.1 M phosphate buffered (saline) solution (PBS) 3 times for 10 min at room temperature, incubated for 90 min in blocking solution consisting of 5% newborn calf serum, and 0.2% Triton in 0.1 M PBS, incubated for 12 h at 4°C in primary antibody (Table I) diluted in blocking solution, incubated at room temperature for 3 h in secondary antibody (Table I) diluted in blocking solution, and counterstained with 49,6-diamidino-2-phenylindole (DAPI, Table I) before being coverslipped with immunofluorescence mounting medium. Slides were washed thoroughly in PBS 4 times between each incubation step. The tissue was imaged with a standard epifluorescence microscope and a confocal microscope. Hematoxylin and eosin staining was done using standard protocols. All tissues were examined histologically after routine H&E staining, and only explants with attached retinas were processed for immunohistochemistry.
Dose-dependence of lentiviral transduction To examine the relationship of transduction efficiency to viral dose, retinal explants were incubated for 24 h with different con-
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centrations of lentiviral vector, followed by 24 h in culture media only. The concentrations of the viral vector used were: 5 3 107 TU, 5 3 108 TU or 5 3 109 TU added to 300 mL of media of 2 explants originating from mice in the 2 different age groups. Expression in the retinal explants was demonstrated by immunohistochemically labelling for the V5-epitope and compared on the basis and distribution of the stain.
Assessment of viability and data analysis To determine the viability of posterior segment explants over time, multiple parameters were assessed and compared to explants from different culture periods, as well as healthy eyes fixed immediately after enucleation. Four explants for each time point (0 to 7 d ex vivo) were used. Explant sections were counterstained with the nuclear marker DAPI. Five sections of each explant were evaluated. Micrographs obtained with a confocal microscope were processed using computer software (Imaris; Leica Microsystems Inc., Concord, Ontario). Creating a 3-dimensional image of the retinal layers allowed an accurate assessment (Figure 2). Nuclei counts were done electronically within a defined volume of each retinal layer. Two cell counts for each of the 4 sections assessed for each explant were obtained and averaged. To avoid variant results caused by regional differences of more peripheral versus central retinal sections within every single explant, central segments as well as peripheral segments were evaluated equally. Sections were stained for activated caspase-3, as previously described. Immunoreactive cells were quantified and compared to the total amount of nuclei. Data were obtained and averaged. For each of the above parameters, changes over time within groups as well as differences between groups at each time point were compared using analysis of variance (ANOVA) with post hoc Scheffe corrections for multiple comparisons. All data are expressed as the mean 6 standard error (SE). A P-value of , 0.05 was considered statistically significant.
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30 mm
Figure 2. Three-dimensional reconstruction of confocal microscopy of a retinal section counterstained with the nuclear marker DAPI, displaying the ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL).
Figure 3. Photomicrograph of a hematoxylin and eosin stained retinal explant at day 2 ex vivo at 103 (A) and 403 (B) magnification, displaying the retinal layers and adjacent choroid and sclera.
Results Preservation of retinal architecture and cell survival Retinal detachment from the RPE was detected macroscopically in one of the samples by day 1. This sample was excluded from the study and replaced with another explant. Staining with H&E in all samples included in the study revealed no RPE hypertrophy in any of the sections examined (Figure 3). To assess the preservation of the retinal microarchitecture over time, we examined DAPI stained explant sections. Overall, the laminar structure of the retina was well-preserved in the posterior
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segment explants of all culture time periods. The DAPI staining also facilitated counting of the nuclei within each retinal layer. Clinically normal control eyes that were fixed whole by immersion in PFA after enucleation, demonstrated normal linear nuclei density. Nuclear counts for the inner nuclear layer (INL) and outer nuclear layer (ONL) were relatively stable until day 7 ex vivo with no significant nuclear loss over time when compared to day 1 ex vivo, whereas nuclei in the retinal ganglion cell layer (GCL) were significantly decreased by day 5 ex vivo. These differences between cell layers were statistically significant. Minimal variability existed among explants culture for the same time period from mice of the same age group as well as different sections of one explant, so micrographs presented are representative of the group analyzed (Figure 4).
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Figure 4. A — Percentage of nuclei within the ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL), compared to clinically normal eyes in vivo (equalling 100%). With the exception of day 0, for each day ex vivo the values represent means of 16 counts: from 4 slides prepared from each of 4 mice. For day 0, 4 slides were prepared from 2 mice. Bars represent standard deviation from the mean. B — Photomicrographs of retinal explant sections counterstained with 49,6-diamidino-2-phenylindole (DAPI) representative of days 1, 4, and 7 ex vivo.
The level of apoptosis within posterior segment explant retinal cells was detected by immunolabelling the sections for activated caspase-3. In freshly isolated retinas no apoptotic labelled cells were detected, which represents the normative data of 0% apoptosis at the start of culture. The level of staining of explants was initially similar between cell layers, with only sporadically (, 10%) occurring positive cells. Over time, caspase staining in the retinal GCL of explants increased significantly (Figure 5).
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Transduction efficiency of retinal explants Transduced cells were detected in both the retinal ONL as well as the INL and GCL in all explants infected with the viral vector construct. No vector associated green fluorescent protein was found in the RPE. Positively stained cells were evenly distributed throughout the explant; however, transduction efficiency was consistently higher in the ONL than in the INL or GCL. Percentage of positive cells in
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Older age group
5 3 107 5 3 108 Figure 5. Percentage of apoptotic cells compared to the absolute nuclei count per area in the different retinal cell layers [ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL)] over the culture period days 0 to 7 ex vivo. Values represent mean counts for each layer from eye cups from 2 different mice. Bars represent standard deviation from the mean.
5 3 109
Younger age group
the different cell layers increased proportionally with increasing vector load. Comparing transduction efficiency of explants from both age groups, a significantly higher number of positive cells was consistently detected in explants of mice euthanized at 4 wk of age compared with those sacrificed at 12 wk of age. The averaged transduction efficiency in the cell layers of all explants is displayed in Figure 6.
Discussion We have developed and characterized a novel technique to culture posterior segment explants that demonstrates tissue viability for approximately 1 wk after enucleation, although ganglion cell apoptosis is noted from 4 d onward. The procedure is relatively easy to perform, provided good quality microsurgical instruments are used to dissect the posterior segments of the globes. The model has several technical advantages, including ease of use and minimal preparation time. Two independent posterior segment explants can be prepared from each animal, thereby reducing the number of animals needed for each experiment. Culture media needs to be exchanged daily in order to maintain the cultures. In this system, conditions can be controlled much more precisely than within live animal models. In addition, explant systems are devoid of immune activity. The use of serum-free media has the advantage of clearly defined ingredients and thus any components that may affect the transduction can be manipulated. Gene transfer to posterior segment explants offers a good method to examine the transduction and expression characteristics of viral vector systems. The method is not suitable for long-term culture (greater than 1 wk). The metabolic needs of the retina, RPE, choroid, and optic nerve once removed from its blood supply exceed that provided by culture media and these sensitive cells die by apoptosis within a few days. Loss of blood circulation reduces oxygen and nutrient supply and cellular waste removal; these metabolic needs are met
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5 3 107 5 3 108
5 3 109
Figure 6. The 5 3 108 or 5 3 109 transduction units of vector expressing green fluorescent protein (GFP). Percentage of cells expressing the transduced gene compared to absolute numbers of cells per layer. Transduction efficiency was consistently higher in retinal cells of mice of the younger age group than the older age group. Note that the transduction efficiency was significantly higher in the outer nuclear layer (ONL) than the inner nuclear layer (INL) or ganglion cell layer (GCL) in both age groups.
by diffusion through the explant tissues from the culture media. We demonstrated that the ganglion cells were lost within 4 d, while the INL and ONL were maintained longer. The 4-day window is adequate to allow for expression of transduced genes and this method allows for a controlled environment to study the role of various factors in retinal gene transfer and to test hypotheses that are difficult to test in vivo. During the 7 d in culture we did not observe gliosis or RPE hypertrophy, which reportedly occur when the neural retina is detached from RPE cells or in culture if the neural retina does not adhere to the substratum (11,16,58,59). The culture of murine posterior segment ocular explants provided viable retinal cell layers for 4 d based on the lack of positive caspase staining. The RPE-choroid-scleral attachment appears to delay some cellular death by maintaining cellular interfaces of RPE and outer
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segments. The apoptosis noted in the ganglion cells was not unexpected as the axonal flow through the optic nerve was disrupted when the eyes were enucleated. Although positive results from in vitro experiments such as this must be followed with live animals studies, our retinal explant culture model is useful for screening therapeutic medications for beneficial effects, assaying the optimum vector concentrations to achieve the desired transduction within target cells, and evaluating the most appropriate age of the animals used. This combined approach could ultimately facilitate reduction and refinement in the number of in vivo procedures required.
Acknowledgment Supported by the Heather Ryan and David Dubé Equine Health Research Fund.
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Development of a murine ocular posterior segment explant culture for the study of intravitreous vector delivery Nora Denk, Vikram Misra, Lynne S. Sandmeyer, Bianca B. Bauer, Jaswant Singh, George W. Forsyth, Bruce H. Grahn
Abstract The objective of this study was to develop a murine retinal/choroidal/scleral explant culture system to facilitate the intravitreous delivery of vectors. Posterior segment explants from adult mice of 2 different age groups (4 wk and 15 wk) were cultured in serum-free medium for variable time periods. Tissue viability was assessed by gross morphology, cell survival quantification, activated caspase-3 expression, and immunohistochemistry. To model ocular gene therapy, explants were exposed to varying transducing units of a lentiviral vector expressing the gene for green fluorescent protein for 48 h. Explant retinal cells remained viable for approximately 1 wk, although the ganglion cell layer developed apoptosis between 4 and 7 d. Following vector infusion into the posterior segment cups, viral transduction was noted in multiple retinal layers in both age groups. An age of donor mouse influence was noted and older mice did not transduce as well as younger mice. This explant offers an easily managed posterior segment ocular culture with minimum disturbance of the tissue, and may be useful for investigating methods of enhancing retinal gene therapy under controlled conditions.
Résumé L’objectif de la présente étude était de développer un système murin de culture d’explant de rétine/choroïde/sclérotique afin de faciliter la livraison intra-vitréenne de vecteurs. Des explants de segments postérieurs provenant de souris adultes de deux groupes d’âge différents (4 sem et 15 sem) furent cultivés dans un milieu sans sérum pour des périodes de temps variables. La viabilité tissulaire fut évaluée par morphologie macroscopique, quantification de la survie cellulaire, expression de la caspase-3 activée, et immunohistochimie. Afin d’imiter la thérapie génique oculaire, les explants furent exposés pendant 48 h à des unités de transduction variables d’un vecteur lentiviral exprimant le gène pour la protéine fluorescente verte. Les explants de cellules de la rétine sont demeurés viables pour environ 1 sem, bien que dans la couche de cellules ganglionnaires on nota le développement de l’apoptose entre 4 à 7 j. Suite à l’infusion de vecteur dans le segment postérieur, la transduction virale fut notée dans plusieurs couches rétiniennes des animaux des deux groupes d’âge. Une influence de l’âge de la souris donneuse fut notée et chez les souris plus âgées la transduction ne se faisait pas aussi bien que chez les jeunes souris. Ce modèle d’explant permet la gestion facile de culture de segment oculaire postérieur avec un minimum de dérangement du tissu, et pourrait être utile pour étudier des méthodes visant à augmenter la thérapie génique sous conditions contrôlées. (Traduit par Docteur Serge Messier)
Introduction Retinal explant culture has been used to study retinal development (1–15), pathophysiological processes of neurodegenerative disease (16–19), central nervous system regeneration (8,13,20–23), cell death and neuroprotection (24–26), electrophysiological recording (27,28), cell transplantation (29–34), test therapeutic substances (16,18,35), examine the role of growth factors (36,37), gene therapy (38), and compare the transduction efficiency of adeno-viral vector mediated gene transfer at different ages in normal mice and mice with retinal degeneration (39). Such studies have involved tissue from embryonic, neonatal animals or from young animals, all of which demonstrate a relatively high level of receptiveness to genetic manipulation compared with adult tissue. We are not aware of any
study that directly compared transduction efficiency in murine posterior ocular explants from 2 age groups. Retinal explant cultures in the first half of the 20th century were primarily grown in plasma clots or in a collagen matrix using a roller tube method, also known as the flying coverslip method, and variations of this method are still used today (11,40–42). In the 1950s, Trowell developed the membrane culture in which the tissue is placed on a porous membrane on top of a wire grid and maintained at the air-medium interface (43). Caffe, et al (44) developed a method in which the neural retina is placed with the vitreous surface facing upwards on rafts made of nitrocellulose filters and polyamide gauze grids. The first retina–retinal pigment epithelium (RPE) culture was reported by Tamai et al (45), and a number of other investigators
Department of Small Animal Clinical Sciences (Denk, Sandmeyer, Bauer, Grahn); Veterinary Microbiology (Misra); and Veterinary Biomedical Sciences (Singh, Forsyth), Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan. Address all correspondence to Dr. Bruce H. Grahn; e-mail: [email protected] Received April 1, 2013. Accepted February 25, 2014. 2015;79:31–38
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Figure 1. This illustration depicts the surgical preparation of freshly enucleated mouse eyes that were used for viral transduction.
have published similar studies of neural retina-RPE cultures from rodents (10,44,46), chickens (47,48), frogs (49,50), and pigs (51). In recent years there have been a number of reports on the culture of isolated neural retina that is not attached to the RPE and choroid. A reduction of apoptosis in retina–RPE culture, compared to cultures of isolated retina culture has been reported (52). Others have described an explant model of whole globes punctured with a needle to enable fluid exchange, although tissue viability was not reported (52,53). Despite promising results in retinal explant culture, the viability and behavior of retinal tissue from adult rodents has not been rigorously evaluated. The integrity of the retina and the ability to genetically modify cells that remain intact are important if this model is to be used in gene therapy development. Intra-vitreous delivery of medications and therapies for acquired and developmental ocular diseases has become a common ophthalmological examination and operating room procedure during the last decade. The development of gene therapy is in its embryologic stages and future development will benefit from coordination of cell culture models that allow for the optimization of vitreous delivery. Posterior segment culture models that facilitate expression/concentration/and vector comparison data, allow for alternative delivery systems, and adjunctive medication strategies prior to in vivo animal model trials are paramount. These techniques will not replace the use of in vivo models; however, they may improve efficiency and reduce the numbers of animals utilized in the future. The purpose of this study was to establish cultures of retina-RPEchoroid-sclera explants from mice of different ages, to define their survival in culture, and to compare transduction efficiency.
Materials and methods Animals Adult c57Bl/6 mice, referred to as wildtype, were obtained from the local animal resource facility. All animals were maintained, treated, and sacrificed in compliance with the Canadian Council of Animal Care Guide to the Care and Use of Experimental Animals, and the University of Saskatchewan Animal Care Committee approved the research protocol. Only mice with eyes that were devoid of any ocular abnormalities based on biomicroscopic and indirect ophthalmoscopic examinations were utilized. Sixteen mice were sacrificed at 2 different postnatal ages (4 and 15 wks of age) by intraperitoneal injection of pentobarbital (100 mg/kg BW), the eyes immediately enucleated postmortem and posterior segment explants were prepared.
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Lentiviral vector system The green fluorescent protein (GFP) coding sequences were amplified by polymerase chain reaction (PCR) from the plasmid pcDNA3.1-GFP and the nucleotide sequence was confirmed by DNA sequencing. The PCR product was cloned into pCR 8-TOPO using a cloning kit (TOPO cloning kit; Life Technologies Inc, Burlington, Ontario) and then transferred to pLenti6.3 (Lentivirus Kit; Life Technologies) using another cloning kit (Gateway cloning kit; Life Technologies). A V5 epitope enables immunolabelling of the gene product. Lentiviral vector was produced following the manufacturer’s instructions using modified and optimized ratios of reagents (Invitrogen, Carlsbad, California, USA). The titer of the virus stock was assessed by transduction of HEK 293 and Vero cells in vitro, followed by immunofluorescence and expressed as transducing units (TU). Manipulations of lentiviral vectors were done in accordance with institutional and national biosafety restrictions and a biosafety permit issued by the University of Saskatchewan Biosafety Committee.
Posterior segment explant preparation and culture Immediately following euthanasia both eyes were enucleated and rinsed with ice-cold balanced salt solution (BSS) containing penicillin (100 U/mL) and streptomycin (100 g/mL). All preparation steps were performed at room temperature. With the aid of an operating microscope scleral incisions were made using 6500 Beaver blades (Lab Tician, Oakville, Ontario) approximately 1 to 2 mm posterior to the limbus of each eye (Figure 1). The stab incision was extended circumferentially with micro scissors to separate the anterior segment of each globe, including the lens and the attached vitreous from the remaining posterior segment. The retina — RPE — choroid — scleral explants were placed in cell culture dishes containing 300 mL of prewarmed media with the inner limiting membrane side facing up (Figure 1). Medium consisted of L-glutamine (0.8 mM) containing neuronal growth medium (Neurobasal A; Invitrogen Ltd.), supplemented with 2% B27 (Invitrogen Ltd.), 1% N2 (Invitrogen Ltd.), penicillin (100 IU/mL), and streptomycin (100 g/mL) as previously described (54). Posterior segment explant cultures were maintained in humidified incubators at 37°C and 5% CO2 for periods of 2 to 7 d. Half of the media were changed every following day. Each culture was repeated 4 times with tissues originating from 4 different mice from 2 different age groups (4 wk and 15 wk of age). Four eyes from age-matched mice that were examined and then euthanized, as previously described, were enucleated and fixed in chilled 4% paraformaldehyde (PFA) and these tissues served as controls. Culture of
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Table I. Details of antibodies and fluorescent markers used for immunohistochemistry Source Specificity Cells labeled (isotype) Conjugate Clone Company Primary antibodies Active caspase-3 Apoptotic cells Rabbit NA Polyclonal Abcam, Cederlane, Burlington, Ontario
Working concentration (mg/mL)
2.0
V5 V5-epitope Mouse NA Polyclonal
Invitrogen, Carlsbad, California, USA
5.0
Secondary antibodies and other labels Rabbit IgG (H 1 L)
NA
Goat
AlexaFluor 488
NA
Invitrogen
2.0
Rabbit IgG (H 1 L)
NA
Goat
AlexaFluor 546
NA
Invitrogen
2.0
Mouse IgG (H 1 L)
NA
Donkey
AlexaFluor 633
NA
Abcam, Cederlane
2.0
Mouse IgG (H 1 L)
NA
Goat
AlexaFluor 488
NA
Invitrogen
2.0
NA
Invitrogen
0.5
DAPI NA Ig — immunoglobulin; DAPI: 49,6-diamidino-2-phenylindole; NA — Not available.
2 whole globes incised only with a perilimbal incision but without separation of the anterior segment was done.
Tissue processing Tissues were fixed by replacing the culture media with freshly prepared 4% PFA for 12 h at 4°C. Whole eyes were enucleated from mice after euthanasia and immersion-fixed in 4% PFA for 12 h at 4°C. The explants and whole eyes were then cryoprotected in 30% sucrose for 24 h at 4°C before being embedded in optimal cutting temperature (OCT) compound (Sakura Finetek USA Inc., Torrance, California, USA), and frozen at 220°C. Posterior segment explants were sectioned vertically [20 mm thickness for immunohistochemistry, 6 mm thickness for hematoxylin and eosin (H&E) staining] on a cryostat microtome at 220°C. For immunohistochemistry, serial sections were mounted on poly-l-lysine coated slides and stored at 220°C. Slides were the washed in 0.1 M phosphate buffered (saline) solution (PBS) 3 times for 10 min at room temperature, incubated for 90 min in blocking solution consisting of 5% newborn calf serum, and 0.2% Triton in 0.1 M PBS, incubated for 12 h at 4°C in primary antibody (Table I) diluted in blocking solution, incubated at room temperature for 3 h in secondary antibody (Table I) diluted in blocking solution, and counterstained with 49,6-diamidino-2-phenylindole (DAPI, Table I) before being coverslipped with immunofluorescence mounting medium. Slides were washed thoroughly in PBS 4 times between each incubation step. The tissue was imaged with a standard epifluorescence microscope and a confocal microscope. Hematoxylin and eosin staining was done using standard protocols. All tissues were examined histologically after routine H&E staining, and only explants with attached retinas were processed for immunohistochemistry.
Dose-dependence of lentiviral transduction To examine the relationship of transduction efficiency to viral dose, retinal explants were incubated for 24 h with different con-
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centrations of lentiviral vector, followed by 24 h in culture media only. The concentrations of the viral vector used were: 5 3 107 TU, 5 3 108 TU or 5 3 109 TU added to 300 mL of media of 2 explants originating from mice in the 2 different age groups. Expression in the retinal explants was demonstrated by immunohistochemically labelling for the V5-epitope and compared on the basis and distribution of the stain.
Assessment of viability and data analysis To determine the viability of posterior segment explants over time, multiple parameters were assessed and compared to explants from different culture periods, as well as healthy eyes fixed immediately after enucleation. Four explants for each time point (0 to 7 d ex vivo) were used. Explant sections were counterstained with the nuclear marker DAPI. Five sections of each explant were evaluated. Micrographs obtained with a confocal microscope were processed using computer software (Imaris; Leica Microsystems Inc., Concord, Ontario). Creating a 3-dimensional image of the retinal layers allowed an accurate assessment (Figure 2). Nuclei counts were done electronically within a defined volume of each retinal layer. Two cell counts for each of the 4 sections assessed for each explant were obtained and averaged. To avoid variant results caused by regional differences of more peripheral versus central retinal sections within every single explant, central segments as well as peripheral segments were evaluated equally. Sections were stained for activated caspase-3, as previously described. Immunoreactive cells were quantified and compared to the total amount of nuclei. Data were obtained and averaged. For each of the above parameters, changes over time within groups as well as differences between groups at each time point were compared using analysis of variance (ANOVA) with post hoc Scheffe corrections for multiple comparisons. All data are expressed as the mean 6 standard error (SE). A P-value of , 0.05 was considered statistically significant.
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30 mm
Figure 2. Three-dimensional reconstruction of confocal microscopy of a retinal section counterstained with the nuclear marker DAPI, displaying the ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL).
Figure 3. Photomicrograph of a hematoxylin and eosin stained retinal explant at day 2 ex vivo at 103 (A) and 403 (B) magnification, displaying the retinal layers and adjacent choroid and sclera.
Results Preservation of retinal architecture and cell survival Retinal detachment from the RPE was detected macroscopically in one of the samples by day 1. This sample was excluded from the study and replaced with another explant. Staining with H&E in all samples included in the study revealed no RPE hypertrophy in any of the sections examined (Figure 3). To assess the preservation of the retinal microarchitecture over time, we examined DAPI stained explant sections. Overall, the laminar structure of the retina was well-preserved in the posterior
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segment explants of all culture time periods. The DAPI staining also facilitated counting of the nuclei within each retinal layer. Clinically normal control eyes that were fixed whole by immersion in PFA after enucleation, demonstrated normal linear nuclei density. Nuclear counts for the inner nuclear layer (INL) and outer nuclear layer (ONL) were relatively stable until day 7 ex vivo with no significant nuclear loss over time when compared to day 1 ex vivo, whereas nuclei in the retinal ganglion cell layer (GCL) were significantly decreased by day 5 ex vivo. These differences between cell layers were statistically significant. Minimal variability existed among explants culture for the same time period from mice of the same age group as well as different sections of one explant, so micrographs presented are representative of the group analyzed (Figure 4).
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Figure 4. A — Percentage of nuclei within the ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL), compared to clinically normal eyes in vivo (equalling 100%). With the exception of day 0, for each day ex vivo the values represent means of 16 counts: from 4 slides prepared from each of 4 mice. For day 0, 4 slides were prepared from 2 mice. Bars represent standard deviation from the mean. B — Photomicrographs of retinal explant sections counterstained with 49,6-diamidino-2-phenylindole (DAPI) representative of days 1, 4, and 7 ex vivo.
The level of apoptosis within posterior segment explant retinal cells was detected by immunolabelling the sections for activated caspase-3. In freshly isolated retinas no apoptotic labelled cells were detected, which represents the normative data of 0% apoptosis at the start of culture. The level of staining of explants was initially similar between cell layers, with only sporadically (, 10%) occurring positive cells. Over time, caspase staining in the retinal GCL of explants increased significantly (Figure 5).
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Transduction efficiency of retinal explants Transduced cells were detected in both the retinal ONL as well as the INL and GCL in all explants infected with the viral vector construct. No vector associated green fluorescent protein was found in the RPE. Positively stained cells were evenly distributed throughout the explant; however, transduction efficiency was consistently higher in the ONL than in the INL or GCL. Percentage of positive cells in
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Older age group
5 3 107 5 3 108 Figure 5. Percentage of apoptotic cells compared to the absolute nuclei count per area in the different retinal cell layers [ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL)] over the culture period days 0 to 7 ex vivo. Values represent mean counts for each layer from eye cups from 2 different mice. Bars represent standard deviation from the mean.
5 3 109
Younger age group
the different cell layers increased proportionally with increasing vector load. Comparing transduction efficiency of explants from both age groups, a significantly higher number of positive cells was consistently detected in explants of mice euthanized at 4 wk of age compared with those sacrificed at 12 wk of age. The averaged transduction efficiency in the cell layers of all explants is displayed in Figure 6.
Discussion We have developed and characterized a novel technique to culture posterior segment explants that demonstrates tissue viability for approximately 1 wk after enucleation, although ganglion cell apoptosis is noted from 4 d onward. The procedure is relatively easy to perform, provided good quality microsurgical instruments are used to dissect the posterior segments of the globes. The model has several technical advantages, including ease of use and minimal preparation time. Two independent posterior segment explants can be prepared from each animal, thereby reducing the number of animals needed for each experiment. Culture media needs to be exchanged daily in order to maintain the cultures. In this system, conditions can be controlled much more precisely than within live animal models. In addition, explant systems are devoid of immune activity. The use of serum-free media has the advantage of clearly defined ingredients and thus any components that may affect the transduction can be manipulated. Gene transfer to posterior segment explants offers a good method to examine the transduction and expression characteristics of viral vector systems. The method is not suitable for long-term culture (greater than 1 wk). The metabolic needs of the retina, RPE, choroid, and optic nerve once removed from its blood supply exceed that provided by culture media and these sensitive cells die by apoptosis within a few days. Loss of blood circulation reduces oxygen and nutrient supply and cellular waste removal; these metabolic needs are met
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5 3 107 5 3 108
5 3 109
Figure 6. The 5 3 108 or 5 3 109 transduction units of vector expressing green fluorescent protein (GFP). Percentage of cells expressing the transduced gene compared to absolute numbers of cells per layer. Transduction efficiency was consistently higher in retinal cells of mice of the younger age group than the older age group. Note that the transduction efficiency was significantly higher in the outer nuclear layer (ONL) than the inner nuclear layer (INL) or ganglion cell layer (GCL) in both age groups.
by diffusion through the explant tissues from the culture media. We demonstrated that the ganglion cells were lost within 4 d, while the INL and ONL were maintained longer. The 4-day window is adequate to allow for expression of transduced genes and this method allows for a controlled environment to study the role of various factors in retinal gene transfer and to test hypotheses that are difficult to test in vivo. During the 7 d in culture we did not observe gliosis or RPE hypertrophy, which reportedly occur when the neural retina is detached from RPE cells or in culture if the neural retina does not adhere to the substratum (11,16,58,59). The culture of murine posterior segment ocular explants provided viable retinal cell layers for 4 d based on the lack of positive caspase staining. The RPE-choroid-scleral attachment appears to delay some cellular death by maintaining cellular interfaces of RPE and outer
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segments. The apoptosis noted in the ganglion cells was not unexpected as the axonal flow through the optic nerve was disrupted when the eyes were enucleated. Although positive results from in vitro experiments such as this must be followed with live animals studies, our retinal explant culture model is useful for screening therapeutic medications for beneficial effects, assaying the optimum vector concentrations to achieve the desired transduction within target cells, and evaluating the most appropriate age of the animals used. This combined approach could ultimately facilitate reduction and refinement in the number of in vivo procedures required.
Acknowledgment Supported by the Heather Ryan and David Dubé Equine Health Research Fund.
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