The subretinal implantation of retinal pigmented epithelium (RPE) is one of the most promising approaches for the treatment of degenerative retinal diseases. However, the performance of preclinical studies on large-eye animal models remains challenging. This report presents guidelines for the subretinal transplantation of RPE on a cell carrier into minipigs.
Degenerative disorders of the retina (including age-related macular degeneration), which originate primarily at or within the retinal pigmented epithelial (RPE) layer, lead to a progressive disorganization of the retinal anatomy and the deterioration of visual function. The substitution of damaged RPE cells (RPEs) with in vitro cultured RPE cells using a subretinal cell carrier has shown potential for re-establishing the anatomical structure of the outer retinal layers and is, therefore, being further studied. Here, we present the principles of a surgical technique that allows for the effective subretinal transplantation of a cell carrier with cultivated RPEs into minipigs. The surgeries were performed under general anesthesia and included a standard lens-sparing three-port pars plana vitrectomy (PPV), subretinal application of a balanced salt solution (BSS), a 2.7 mm retinotomy, implantation of a nanofibrous cell carrier into the subretinal space through an additional 3.0 mm sclerotomy, fluid-air exchange (FAX), silicone oil tamponade, and closure of all the sclerotomies. This surgical approach was used in 29 surgeries (18 animals) over the past 8 years with a success rate of 93.1%. Anatomic verification of the surgical placement was carried out using in vivo fundus imaging (fundus photography and optical coherence tomography). The recommended surgical steps for the subretinal implantation of RPEs on a carrier in minipig eyes can be used in future preclinical studies using large-eye animal models.
Age-related macular degeneration (AMD) is considered the main cause of central vision loss in developed countries and is one of many conditions related to retinal pigmented epithelium (RPE) dysfunction1,2. The RPE is found on the basally located Bruch's membrane (BM) and provides the necessary maintenance for the photoreceptors. The progressive degeneration of the RPE layer is a hallmark of the early atrophic form of AMD, and it also accompanies the development of the late exudative form of AMD as well. Despite many advances in retinal disease therapy, the development of an effective treatment modality remains challenging3. One of the promising methods is RPE replacement using an in vitro cultured RPE layer. This treatment is associated with progress in stem cell research using human embryonic stem cell-derived RPE (hESC-RPE) and induced pluripotent stem cell-derived RPE (iPSC-RPE)3,4,5,6,7. In recent years, many research groups have focused on developing different approaches for RPE replacement with the initially accepted proof-of-concept8,9,10,11,12,13,14,15. The RPE cells (RPEs) are usually delivered into the subretinal space in the form of a cell suspension, a self-supporting cell sheet, or a cell monolayer supported by an artificial carrier3,16,17,18,19,20,21. The injection of a cell suspension is the easiest method, but the compromised condition of the BM can often prevent the attachment of the transplanted cells. This can result in incorrect apicobasal orientation of the RPEs and failure to form a monolayer22,23. The main advantage of the other two methods (i.e., a self-supporting cell sheet and a cell monolayer supported by an artificial substrate) is that the cells are already in a differentiated monolayer state when implanted directly into the subretinal space24.
Many surgical techniques describing the delivery of cell carriers into the subretinal space have been published in recent years8,9,10,11,12,13,14,15. These studies described the use of large-eye animal models, the types of cellular carriers, the use of transplanted cellular cultures, the implantation instruments, as well as the surgical techniques, and the authors focused mainly on the results of subretinal implantation. In 2015, Popelka et al. reported the use of a frame-supported ultrathin electrospun polymer membrane for the transplantation of RPEs into porcine cadaver eyes8. The surgical technique described here with subretinal implantation of the cell carrier allowed for relatively precise handling of the carrier and easy positioning of the scaffold in the subretinal space. Kozak et al. assessed the feasibility of the delivery technique of a carrier with an approximate size of 2 mm x 5 mm in porcine eyes9. The unique design of the cell carrier permitted its correct placement, preventing the cellular monolayer from folding and wrinkling6. Al-Nawaiseh et al. first presented detailed step-by-step guidelines for subretinal scaffold implantation in rabbits25. Stanzel et al. then published a similar protocol in 2019 for transplantation in small rodents, rabbits, pigs, and nonhuman primates26. As published previously, the transplantation of a differentiated and polarized RPE monolayer on a solid carrier resulted in improved survival and better integration of the graft in comparison to other delivery techniques (Supplementary File 1)27.
The purpose of any preclinical animal studies performed in vivo is to reveal the various aspects of surgical transvitreal subretinal implantation of a cell carrier with a focus on the procedure safety, the survival of the transplanted cells, the tissue response to the subretinal maneuvers, and the short- and long-term postoperative outcomes. The use of porcine eyes as a large-eye animal model has been reported to be relevant in terms of the scope of the data obtained, which could be useful and potentially applicable to humans10,11,14. Our study reports the surgical technique used for the in vivo subretinal implantation of a cell carrier in a large-eye animal model. We present a detailed description of the preoperative preparations, the surgical technique of subretinal cell carrier implantation, and the postoperative care of the minipig eyes based on our experience over the last 8 years. We describe the basic surgical principles that can be used for in vivo experimental studies involving the implantation of different types of cells and cell carriers.
Large animal model
The experimental herd of Liběchov minipigs was founded by importing five animals from the Hormel strain from the USA in 1967. These animals were crossbred for porcine blood group studies with several other breeds or strains: Landrace, Large White, Cornwall, Vietnamese pigs, and miniature pigs of Göttingen origin28,29. At 5 months of age and approximately 20 kg body weight (BW), the minipigs reach sexual maturity. The survival of the parental minipig breeds (Hormel and Göttingen) is reported to be 12-20 years. The subretinal implantation of the cell carrier targets the central portion of the retina. The retina of minipigs lacks a macula and fovea. However, it has regions of highly concentrated cone photoreceptors called the area centralis and visual streaks30,31. These regions are responsible for the highest visual acuity.
The surgeries were performed by four experienced vitreoretinal surgeons with the assistance of an experienced surgical facility assistant (TA). Before the in vivo experiments, the surgeons were educated and obtained special knowledge of minipig eye anatomy, such as regarding the lower ratio of lens to vitreous volume, the shorter axial length (15-19 mm), the absence of the Bowman's membrane in the cornea, the smaller vitreous volume (2.8-3.2 mL), the absence of the macula and fovea, the absence of the annulus of Zinn, and the optic disc diameter (vertical/horizontal: 1.5 mm/2.1 mm). In all cases, the surgery was performed under general anesthesia in a specially organized operating room with the implementation of standard aseptic and antiseptic measures.
This study adheres to the tenets of the Guidelines of the Declaration of Helsinki and ethical principles for medical research involving human subjects. All experiments were carried out according to the Guidelines for the Care and Use of Laboratory Animals and according to the Association for Research in Vision and Ophthalmology (ARVO) for the use of animals in ophthalmic and visual research. The study protocol was approved by the Resort Professional Commission of the CAS for Approval of Projects of Experiments on Animals at the Institute of Animal Physiology and Genetics of the Czech Academy of Sciences (Liběchov, Czech Republic) (Approved protocol No. 60/2016 and No. 64/2019).
1. Considerations during the subretinal transplantation of cells on a carrier into minipigs
Figure 1: Schematic drawing of the retinal zones in minipigs. (A) Schematic drawing of the retinal zones in relation to the minipig's head; the yellow ellipse depicts the desired area of subretinal implantation, T refers to the temporal retinal area, and N refers to the nasal retinal area. (B) Example of the fundus scheme after subretinal implantation of the cell carrier (yellow) through retinotomy (red). Please click here to view a larger version of this figure.
Figure 2: Transportation and placement of the animal. (A) Transportation of the sedated animal to the operating room. (B) Placement of the animal during intubation. (C) Adjustment of the head of the animal for optimal access to the central retina during the surgery (red arrow). Please click here to view a larger version of this figure.
Figure 3: The standard operating room setup. (A) Schematic depiction of the surgeons' position (S = surgeon, A = assistant) in relation to the position of the operating table with the minipig, the operating microscope (OM), the vitrectomy machine (VM), the instrumental table (IT), and the anesthesiology machine (AM). There are two possible positions of the vitrectomy machine (yellow and gray). (B) Real-life setting in the operating room. Please click here to view a larger version of this figure.
2. Cell carrier, cultivated cells cultures, and implantation injector
Figure 4: Nanofibrous carrier with an embedded supporting PET frame. (A) Three visible marks on the frame allow control of the side orientation of the carrier (white arrows). (B) Enlargement view of the PET frame fragment embedded in the nanofibrous membrane (white arrows) of the cell carrier. Please click here to view a larger version of this figure.
Figure 5: Implantation injector. (A) Parts of the injector. (B) Nanofibrous cell carrier with embedded supporting PET frame loaded to the plastic rectangular capillary of the implantation injector. Please click here to view a larger version of this figure.
3. Surgical procedure
Figure 6: Insertion of the trocars in the eye of a minipig. (A) Schematic depiction of the trocars, which are inserted perpendicularly into the sclera toward the center of the vitreous cavity in the human eye (gray color) and in an oblique manner toward the posterior retina in the minipig eye (blue color) to avoid damage to the lens. The lens of the minipig (blue colored) is larger than that in humans and relative to the vitreous cavity size. (B) Intraoperative view of the inserted trocars in a three-port PPV. The cornea is covered with methylcellulose to prevent drying and swelling. Please click here to view a larger version of this figure.
4. Postoperative care
5. Postoperative procedures
6. Enucleation of the eye post mortem after euthanasia
The results of the subretinal implantation of the cell carrier in Liběchov minipigs are presented in Table 2. Successful implantation was defined as obtaining sufficient data for histologic and immunohistochemical study. Failed cases were defined as eyes with severe intraoperative complications, which made further observation of the eye tissues impossible.
The application of the proposed technique with the use of silicone oil tamponade allows for controlling the condition of the subretinal transplant using imaging modalities starting from the next day after the surgery until the time of enucleation (Figure 7, Figure 8, and Figure 9).
Fundus imaging and SD-OCT
The minipigs were examined in the postoperative period using fundus imaging, red-free imaging, and spectral domain optical coherent tomography (Figure 7). High-quality fundus imaging was enabled by using clear optic media, including a clear lens and the use of silicone oil tamponade (Figure 7A). The site of the retinotomy showed no signs of a proliferative reaction (Figure 7A, yellow arrows), and the PTE frame of the cell carrier was clearly visible through the semi-transparent layers of the porcine retina. On the red-free imaging, the reflectivity of the cultivated hRPEs on the carrier did not differ from the reflectivity of the endogenous porcine RPE layer (Figure 7B). On the SD-OCT, the PTE frame caused only minor shadowing of the underlying anatomical structures and slight thickening of the retina (Figure 7C, red arrows). No atypical hypo- or hyper-reflective zones were noticed on the SD-OCT, and the Bruch's membrane appeared to remain undamaged as well. Figure 8 presents fundus and iOCT images of the scaffold cultivated with primary human RPE cells 1 month after the surgery (Figure 8). The cell carrier itself (without any cells) caused no significant increase in retinal thickness (Figure 9C). These findings suggest that the intraoperative iatrogenic impact of the implant was minimal and that the implanted cell carrier underwent sufficient adaptation of the implanted cells to the overlying photoreceptor cells and neuroretinal tissue.
Figure 7: Postoperative imaging of the retina in minipigs. (A) Fundus imaging, (B) Red-free image, and (C) optical coherence tomography imaging of the nanofibrous carrier with primary human RPE cells in a 1 week follow-up after subretinal transplantation in a minipig eye. (A) The yellow arrows indicate the site of retinotomy. (B) Red arrows demonstrate the margins of the nanofibrous cell carrier. (C) The red arrows show the slight shadowing of the OCT signal caused by the supporting PET frame of the nanofibrous carrier, which was implanted into the subretinal space. Please click here to view a larger version of this figure.
Figure 8: Fundus imaging and iOCT images of the scaffolds 30 days after subretinal implantation in the minipigs. A, B, C, D, and E correspond to pigs 169, 182, 179, 199, and 224, respectively. The yellow arrows depict the frame of the scaffold. Please click here to view a larger version of this figure.
Histological and immunohistochemical analysis
After the euthanasia of the animals, whole minipig eyes were removed and fixed in 4% paraformaldehyde (PFA) for 24 h. The anterior part of the eye was removed, and the implanted nanofibrous carrier was identified in the nasal central retina and isolated with the sclera attached. All the tissues were cryoprotected in graded sucrose solutions, and vertical frozen sections were cut, as described in detail43. Histology of the nanofibrous membrane without RPE cells after 4 weeks of implantation revealed retinas without inflammation and degenerative changes (Figure 9A). The presence of the nanofibrous membrane was detected in polarized light (Figure 9B).
Figure 9: Histological analysis of the implanted acellular nanofibrous membrane. Hematoxylin-eosin staining of the acellular nanofibrous membrane 4 weeks after implantation (A) with standard illumination and (B) with polarized light microscopy. The white arrow indicates the nanofibrous membrane localization (scale bar: 50 µm). (C) In vivo optical coherence tomography pictures of the acellular nanofibrous membrane after 4 weeks after implantation depicts good acceptance and adherence of the nanofibrous membrane in the subretinal space. The white arrow indicates the location of the implant in the cross-sectional image of the retina. Please click here to view a larger version of this figure.
Figure 10 shows the hematoxylin-eosin (H&E) staining of the retinal area containing the implanted primary hRPE cells on a nanofibrous carrier (yellow arrow) in the minipig eye. The pigmented appearance of the implanted primary hRPEs formed a continuous yet irregular pigmented layer (Figure 10, red arrows). After longer observation periods (6 weeks), the neuroretina underneath the implants showed a rosette-like or hypertrophic reaction-like appearance around the retinotomy site, likely as a result of iatrogenic manipulation. These morphologic results are comparable to the SD-OCT findings and support the evidence for the minimal impact of carrier delivery on the retinal tissue.
Figure 10: Histological analysis of the implanted nanofibrous membrane with the primary hRPEs. Hematoxylin-eosin staining of the retinal area containing the implanted nanofibrous carrier (yellow arrow) with the primary hRPEs in the minipig eye. The animal was euthanized and analyzed 6 weeks after implantation. The primary hRPEs were clearly distinguishable by their size, round shape, and pigmentation (red arrows) in the subretinal space opposite the photoreceptors. The photoreceptor nuclei in the ONL build rosette-like structures. The subretinal space appears hypertrophic. Abbreviations: hRPE = primary human retinal pigmented epithelium, ONL = outer nuclear layer, INL = inner nuclear layer. Please click here to view a larger version of this figure.
Immunostaining was performed employing a two-step indirect method. The sections were incubated at room temperature overnight in CRALBP, a monoclonal primary antibody, at a dilution of 1:100. Immunofluorescence was performed using Alexa Fluor 488-conjugated secondary antibody.
The implanted primary hRPEs were present in the area of implantation and expressed the typical RPE CRALBP marker similar to the endogenous minipig RPE cells (Figure 11A). In contrast, the morphology of the implanted cells appeared to not assume a monolayer shape after implantation yet remained localized within the defined subretinal space (Figure 11A,B, white arrows). The following RPE/retinal markers and morphological appearance remained positive after the 6 week post-implantation period: the presence of pigment/melanin granules, the end-stage retinal specific neuronal markers for the rod bipolar (PKC-alpha) and the cone photoreceptors (PNA), and the GFAP positivity-a sign of microglial activation.
Figure 11: Immunolabeling with the RPE cell marker CRALBP (cellular retinaldehyde binding protein) in a minipig 6 months after implantation of primary hRPEs. (A) Vertical frozen sections of the treated pig eye were immunolabeled with CRALBP monoclonal antibody (green) and counterstained with DAPI (blue). (B) Single depiction of cell nuclei labeling with DAPI in black and white, as high contrast reveals the round shape of the individual hRPE cells (some shown with white arrows). Abbreviations: hRPE = human retinal pigment epithelium, ONL = outer nuclear layer. Please click here to view a larger version of this figure.
Ocular complications
In total, there were 27 of 29 (93.1%) successfully performed operations. The definition "successfully performed surgeries" was applied to those cases where the operated eye did not show any clinically significant postoperative complications until the time of enucleation that could influence the histological and immunohistochemical study. Reduced transparency of the optical media impacted the postoperative imaging in four cases (13.7%); nonetheless, these eyes were processed with further histologic and immunohistochemical analysis.
Intraoperative peripheral retinal detachment occurred in four cases (13.8%). In two cases, it was managed by aspiration of the subretinal fluid during fluid-gas exchange and the application of laser photocoagulation of the retina in the area of detachment. In the other two cases (6.9%), the retinal detachment was associated with massive retinal and subretinal bleeding, which made implantation of the cell carrier impossible and led to the termination of the surgery and immediate euthanasia of the minipig while on the operating table.
No | Parameters | Standard used settings |
1 | Vitrectomy speed (cutting rate) | up to 20,000 cuts/min |
2 | Venturi pump | 50-180 mmHg |
3 | Rise time | 1 sec |
4 | Irrigation pressure | 18-25 mmHg |
5 | Air infusion pressure | 20-25 mmHg |
6 | Bipolar exodiathermy | 18-26% |
7 | Monopolar endodiathermy | 16-18% |
8 | Laser photocoagulation of the retina, 532 nm | Power 100-150 mW |
Interval 100 ms | ||
Duration 100 ms |
Table 1: Standard parameters used during vitrectomy and laser photocoagulation.
Total animals, n | 18 |
Total eyes, n | 36 |
Operated eyes, n | 29 |
Successful implantation, n | 27 |
Failed cases, n | 2 |
Mean surgery time, min | 57 |
Success rate, % | 93.1 |
Table 2: Results of the standardized surgical technique with subretinal implantation of the cell carrier in Liběchov minipigs between 2016 and 2020.
Supplementary File 1: Summary of the studies dedicated to the subretinal implantation of RPE cells on the cell carrier. Please click here to download this File.
The subretinal implantation of RPE cells with different origins is a very promising trend in eye research for the treatment of retinal degenerative disorders, such as AMD3,4,8,9,10,11,12,13,14,15,25. The main idea of this approach is to substitute the damaged RPEs with healthy RPEs cultured ex vivo (Supplementary File 1)44,45,46,47,48. The use of cell carriers to transplant the cultivated RPE cells represents the most reasonable approach, since the porous membranes maintain the polarized RPE cell layer in the correct orientation with regard to the photosensory layer.
Optimal animal model
A critical step in developing such treatment approaches is the use of the optimal animal model49. In the past, small and large animal models have been used, including rabbits, dogs, pigs, and non-human primates8,9,10,11,12,13,14,15,27,29. In this paper, we propose the use of the Liběchov minipig model and describe the preoperative, surgical, and postoperative steps that enable robust transplantation efficacies. The Liběchov minipig was originally bred about 20 years ago and has been frequently used in biomedical research in the field of neurodegenerative diseases, such as Parkinson's and Huntington's disease29,50. Since the pig possesses a relatively large brain with a blood supply and immunologic response similar to those in humans, it has been used as an animal model for allogeneic transplantation experiments as well51,52,53,54. Even though the retina of the minipigs does not possess a human-like macula and fovea, it contains the area centralis and visual streaks, which are regions of the retina with a high concentration of cone photoreceptors30. The similar size to the human eye, the presence of a cone-enriched central retina, the well-described immune system, and the presence of methods to assess the morphology and function post-surgery are important arguments for the use of this large animal model in the presented study.
Surgical procedure
To the best of our knowledge, there are no standardized and widely accepted surgical techniques for the vitreoretinal transplantation of RPE cells on carriers. One of the key issues of cell replacement therapy is the challenging surgical technique that has a risk of intraoperative and postoperative complications linked to retinal detachment, hypotony, episcleral, choroidal, and/or retinal bleeding, and high intraocular turbulence, which can lead to scaffold damage. Postoperatively, there is a risk of proliferative vitreoretinopathy, endophthalmitis, hypotony, retinal detachment, and cataract formation4,10,13,14,15.
The first studies on approaches using cell carriers were performed in chinchilla bastard rabbits13,16,25. Even though these animals represent a small animal model, the results focusing on the technical aspects of the surgery were crucial in the development of the procedures in large animal models and are, therefore, summarized below.
A custom-made 23 G infusion cannula was initially used with two side ports in order to redirect the jet stream, which helped to resolve the collapse of the bleb and consequent retinal detachment13. In the present study, we did not notice any such collapse of the bleb. The possible reason for that could be the bigger size of the eyeball and the performance of the core vitrectomy with spared vitreous on the periphery at the cannula infusion site, which could reduce the force of the directed jet stream.
Difficulties during the ejection of the cell carrier from the instrument were another intraoperative obstacle in the small animal models, which were categorized as "trapped with the instrument". Additionally, the authors suggested that the residual vitreous on the retinal surface could cause a backward "jump" of the carrier out of the retinotomy orifice after implantation. This problem can be solved with an enzyme-assisted vitrectomy, which enables a smooth, continuous ejection of the cell carrier into the subretinal space. In the majority of cases, the authors repositioned the carrier to obtain a more distant location of the implant away from retinotomy. In our case series, we also experienced a situation in which the cell carrier remained attached to the tip of the injector. However, that was managed by slow and gentle manipulation of the light pipe and the injector's tip. We did not observe any residual vitreous at the site of retinotomy in any of our cases. The use of TA-assisted PPV in the surgeries can be suggested as a method to reduce the risk of residually attached vitreous. Multiple staining with TA may be necessary to remove the overlying vitreous completely.
In a different study, the results of subretinal implantation of human RPE stem cells grown as a polarized cellular monolayer on a polyester membrane were reported24. During the experiments, the same surgical technique described previously was used13, but a two-port PPV approach was applied. Finally, a step-by-step protocol for the subretinal implantation of cell carrier surgery in rabbits was published subsequently25. This study presents a very detailed and easily repeatable description of the surgical procedure, including preoperative and postoperative care, which are based on previous experience as well.
During the use of large animal models in subsequent studies, not only technical questions were addressed but also questions regarding the immune reaction to the transplanted cells, as well as cell carrier size-related issues. A study using cynomolgus monkeys (Macaca fascicularis) described the results of the subretinal implantation of human stem cell-derived RPE monolayers15. All the animals underwent systemic immunosuppression, which consisted of sirolimus (loading dose of 2 mg, daily dose of 1 mg) and tetracycline (7.5 mg/kg– BW) starting 7 days prior to the surgery and lasting 3 months after the surgery. The surgical procedure was performed according to protocols described previously24,25. The authors used a 25 G three-port PPV approach with chandelier endo-illumination. Importantly, a TA-assisted PVD was used to exclude residual vitreoretinal adhesion on the posterior retina. As an addition to the originally described procedure, the authors removed the host RPE layer in the area of future implantation using a 20 G custom-made extendable loop instrument.
In our minipig study, we also used systemic immunosuppression. However, the type of immunosuppression differed from the one described above. We administered a subcutaneous injection of tacrolimus-eluting polymer microspheres as a depot at a dose of 0.25 mg/kg BW to hamper cell graft rejection and inflammatory reactions. We did not remove the host RPE cell layer during surgery, as our primary aim was to analyze the safety of the procedure and the viability of the implanted cells but not their integration into the host retina.
Previously, the safety and feasibility of the subretinal implantation of a monolayer of hESC-derived RPEs on a foldable non-degradable mesh-supported submicron parylene-C membrane (6.25 mm x 3.5 mm, 0.4 µm thick) was assessed in 14 female Yucatán minipigs10. After cultivation, the cells were seeded onto a mesh-supported membrane. Immunosuppression was performed using the systemic administration of tacrolimus (no regime and dose indicated) and intravitreal injections of 0.7 mg of a dexamethasone implant at the end of surgery. PPV was performed with a 20 G approach. The authors used an intravitreal injection of triamcinolone acetonid for better visualization of the vitreous body. The large sclerotomy was 2 mm to 3 mm in size. After the subretinal injection, the retina was flattened with a temporary injection of perfluorocarbon liquid. After the fluid-air exchange, a silicone oil tamponade (1,000/5,000 cSt) was performed. Postoperative care included the ocular application of dexamethasone/neomycin/polymyxin B ointment 1 week after the surgery. The authors reported a success rate of 91% (i.e., efficient subretinal implantation and sufficient postoperative imaging data). In our study, the intravitreal injection of TA crystals was used intraoperatively and mainly to visualize the vitreous body. However, the local immunosuppressive action of this drug remains unclear. The nanofibrous cell carriers used in our study were 5.2 mm x 2.1 mm and 3.7 µm thick, with pore sizes of 0.4 µm. During the surgery, we performed direct FAX instead of injecting perfluorocarbon liquid. Our surgical success rate (93.1%) was consistent with and slightly better than that of Koss et al.10.
The subretinal transplantation of fully degradable cell carriers (scaffold) for subretinal implantation was first studied in 2019 in Yorkshire pigs14. The study was mainly focused on the biodegradable characteristics of fibrin hydrogel implants. The authors noted that the aggressive immunosuppression used on the domestic pigs could inhibit a local inflammatory reaction potentially caused during biodegradation of the fibrin hydrogel implants. However, they did not specify the immunosuppressive therapy used in the pigs. During PPV, they performed a 3.6 mm long sclerotomy for insertion of the subretinal implantation device parallel to and approximately 3.5 mm posterior to the limbus. Additionally, they used a pneumatic-driven injection system aiming to reduce the hand-placement instability caused by finger manipulation. In our case series, all the sclerotomies were 2.5 mm to 3.0 mm from the limbus. The large sclerotomy for the insertion of the injector was 3 mm long. The implantation injector used in our study was operated by hand. Thorough cautery of the pars plana of the ciliary body and a sufficient cut inside the large sclerotomy appear to be crucial for avoiding intraoperative complications such as iatrogenic peripheral retinal detachment, bleeding, and loss of the implant.
In summary, we describe the use of the Liběchov minipig model for the transplantation of RPE cells on biodegradable carriers as a treatment option for inherited and acquired retinal diseases. Similarities in eye anatomy and physiology, as well as with regard to the immune system, allow us to develop and improve on the surgical techniques and instrumentation for the subretinal implantation of cells, which can be easily transferred to the treatment of human eye disorders. It is important to assure that surgeries on minipigs are performed using the same instrumentation (including implantation delivery tools) when utilized in human surgeries, thus facilitating the application of gained experience and know-how to humans. Alternative large-eye animal models with the presence of a macular area, such as non-human primates, could be useful for the follow-up and analysis of the anatomical and functional changes after subretinal implantation in the central retinal area. The detailed description of the preoperative, surgical, and postoperative care procedures will be useful for future studies by increasing efficient and standardized data generation.
The authors have nothing to disclose.
The project was supported by The Czech Science Foundation (Project Number 18-04393S) and Norway Grants and Technology Agency of the Czech Republic (KAPPA Programme, Project Number TO01000107).
Technical equipment | |||
Wato EX-65 with a Mindray iMEC10 | Mindray, Shenzhen, China | Wato Ex-65 | anesthesia machine |
R-Evolution CR | Optikon, Rome, Italy | R-Evolution CR | phacoemulsifier/vitrectome |
Green laser Merilas 532α | Meridian, Thun, Switzerland | Merilas 532α | ophthalmic green laser |
Microscope Hi-R NEO 900A | Haag-Streit Surgical, Wedel, Germany) | Hi-R NEO 900A | ophthalmic surgery microscope |
Camera Sony PMW-10MD | Sony, Tokyo, Japan | PMW-10MD | full HD medical 2-piece |
Non-contact vitreoretinal surgical system MERLIN BIOM | Volk, Mentor, OH, USA | MERLIN BIOM | BIOM |
Steam sterilizer | Tuttnauer Europe B.V., Breda, NL | 3870 HSG | sterilizer |
iCam100 | Optovue, Fremont, CA, USA | iCAM100 | funduscamera |
iVue OCT100-2 | Optovue, Fremont, CA, USA | iVue OCT100-2 | OCT |
Microsurgical instruments and devices | |||
Cook Eye Speculum | Katena, New Jersey, US | K1-5403 | 15mm blades |
Ophthalmology surgical drape | Hylyard, Alpharetta, Georgie, USA | 79304 | 132 x 142cm |
Disposable Two step vitrectomy system. (23 gauge/ 0.6 mm) | DORC, Zuidland, Netherlands | 1272.ED06 | |
Infusion line for 23G / 0.6 mm infusion cannula | DORC, Zuidland, Netherlands | 1279.P | |
knife 2.75mm, IQ Geometry Tm Slit Knife Angled, Bevel Up | Surgical Specialties Corporation, Reading, USA | 72-2761G | |
Extendible 41G subretinal injection needle. (23 gauge / 0.6 mm) | DORC, Zuidland, Netherlands | 1270.EXT | |
Omnifix 3ml Luer Lock Solo siringe | BBraun, Melsungen, Germany | 4617022V | 3ml |
1ml soft-inject Tuberculin | Henke Sass Wolf, Tuttlingen, Germany | 5010.200V0 | 1ml |
8-0 Coated Vicryl | Ethicon, Puerto Rico, USA | J409G | |
Purified Silicone Oil (in syringe) 10 ml | (FCI, Paris, France) | S5.7170 | 1000cSt |
Pinnacle 360 Morris Vertical Scissors 23Ga | Synergetics, O'Fallon, USA | 10.24.23PIN | 23Ga |
Revolution DSP 23Ga ILM forceps | Alcon, Geneva, Switzerland | 706.44 | Griesharber revolution |
23ga Straight Laser Probe | Synergetics, O’Fallon, USA | 55.21.23 | |
FCI Protect 2.0% | FCI Ophthalmics, Paris, France | S5.9100 | viscoelastic |
DK Westcott style Stitch Scissors, Curve | Duckworth & Kent, Hertfordshire, England | 1-501 | Curve |
Pierse Notched Forceps, 0,3mm Straigh | Duckworth & Kent, Hertfordshire, England | 2-100-1E | 0,3mm straigh |
DK Harms-Tubingen Straight Tying Forceps | Duckworth & Kent, Hertfordshire, England | 2-504E | 6mm |
DK Needle Holder, Straigh | Duckworth & Kent, Hertfordshire, England | 3-201 | 9mm straigh |
Medications and solutions | |||
Unitropic 1% gtt. | UNIMED PHARMA spol. s r.o., Bratislava, Slovak republic | tropicamidum 10 mg/ml | eye drops |
Diprophos | Merck Sharp & Dohme B.V., Haarlem, Netherlands | betamethasonum 7 mg/ml | 1ml |
Alcon BSS Irrigation Solution | Alcon, Geneva, Switzerland | balance salt solution (BSS) | 500ml |
Betaisodona | Mundipharma, Cambridge, United Kingdom | povidon-Iodine 1g/10ml | 30ml |
Depo-medrol 120mg | Pfizer, New Yourk, USA | methylprednisolon | 5ml/200mg |
Shotapen | Virbac Carros Cedex, France | benzylpenicillin, dihydrostreptomycin | 250ml |
Flunixin a.u.v. | Norbrook, Newry, Northern Ireland | flunixinum 50,0 mg | 250ml |
Tramal 100MG/2ML | Stada Arzneimittel AG, Bad Vilbel, Deutschland | tramadol | 2ml |
Zoletil 100 | Virbac Carros Cedex, France | tiletamine, zolazepam | 100mg |
Narkeran 10 | Vetoquinol, Magny-Vernois, France | ketamin | 2ml |
Rometar 20mg/ml | Spofa pharmaceutica, Prague, Czech republic | xylazinum | 20mg |
Braunol 75mg/g | B.Braun medical, Prague, Czech republic | povidone iodine | 75mg/g |
Propofol 1% MCT/LCT | Fresenius Kabi, Bad Homburg, Deutschland | propofol | 10mg/1ml |
Isoflurane 100% Inhalation vapour, liquid | Piramal Critical Care Limited, West Drayton, United Kingdom | isoflurane | 100% |
Benoxi gtt. 4mg/1ml | Unimed pharma, Bratislava, Slovakia | oxybuprakaine | 10ml |
Neosynephrin POS 10% gtt. | Ursapharm , Saarbrücken, Deutschland | fenylefrin chloride | 10ml |
Ophthalmo-framykoin 1X5GM | Zentiva a.s., Prague, Czech republic | bacitracin zinc/hydrocortisone acetate/hydrocortisoneacetate/neomycin sulfate | 5mg |
Floxal ung. | Dr. Gerhard Mann Chem.-Pharm. Fabrik, Berlin, Germany | ofloxacin | 0.30% |
Eficur inj. | Hipra, Amer, Spain | ceftiofurum hydrochloridum | 50mg / 1ml |
Draxxin | Zoetis Inc., New Jersey, USA | tulathromycinum | 100mg / 1ml |
Tramal | Stada Arzneimittel AG, Bad Vilbel, Deutschland | tramadoli hydrochloridum | 100mg / 2ml |
Xylapan | Vetoquinol, Magny-Vernois, France | xylazinum | 0.4 mg/kg |
Proparacaine hydrochlorid ophthalmic solution 0,5% | Bausch&Lomb Incorporated Tampa, FL, USA | Proparacaine hydrochlorid | 0.50% |
Prograf | Astellas Pharma, Deerfield, Illinois, USA | Tacrolimus powder | 1mg |
Cell carrier, cultivated cells cultures, and implantation injector | |||
Falcon Cell Culture Inserts | Corning Inc., Kenneburg, ME, USA | 353103 | |
TrypLE Express Enzyme (1X) | Thermo Fisher Scientific, MA, USA | 12604021 | |
DMEM/F-12 | Thermo Fisher Scientific, MA, USA | 11320033 | |
Biolaminin 521 LN (LN521) | BioLamina, Sundbyberg, Sweden | LN521-02 | |
GlutaMAX Supplement | Thermo Fisher Scientific, MA, USA | 35050061 | |
2-Mercaptoethanol | Thermo Fisher Scientific, MA, USA | J66742.0B | |
Penicillin-Streptomycin | Sigma-Aldrich, San Luis, Mi, USA | P4333 | |
CRALBP | Novus Biologicals, Abingdon, UK | NB100-74392 | |
Alexa Fluor 488 | Thermo Fisher Scientific, Germany | 21202 |