In our previous work with high concentration fibrin hydrogels, they were formed by pressing (Gandhi et al., 2019; Gandhi et al., 2020; Gandhi et al., 2018). During the course of our studies, we found that the thickness of pressed gels was highly variable. This was in part due to the rapid initial gelation of fibrin at supra-physiological concentrations. We later found that the initial gelation could be slowed by the addition of select azo-dyes such as trypan blue and Evans blue (Gandhi et al., 2021). Trypan blue is used in vitreoretinal surgery to stain epiretinal membranes and the inner limiting membrane of the retina, so we choose to add trypan blue to our gelation mix. Doing so slowed gelation kinetics enough to allow us to use injection molding rather than pressing to fabricate a larger hydrogel with control over all three dimensions.

To use injection molding, we needed to design a mold that would yield a suitable blank. Minimum criteria for the blank were that it could yield >60 oval shaped doses of 1.5 × 5.0 × 0.2 mm and be in a format that could be utilized in cell culture for >30 days prior to punching/cutting of individual doses. This requires that the hydrogel have mechanical support for >30 days and that the hydrogel blank and support fit in a suitable cell culture vessel. To meet these criteria, it was considered essential, upon opening, that the gel blank remained adhered to the mold cavity, that the mold cavity be on only one side of the mold, that the mold plate not float in aqueous solutions, and that the mechanical support be produced from a material that is biocompatible and could meet ISO 10993 specifications.

Based on extensive prototyping, we settled on a single use disposable mold design (Figures 1A–D) that uses a machined polycarbonate top plate with an 18-gauge tapered inlet port placed perpendicular to the mold. The polycarbonate plate is clear, permitting the operator to observe its filling during fabrication, and unlike polystyrene, the polycarbonate plate does not float in aqueous solution. A set of channels were milled in the mold cavity running from the inlet port to the middle and both outside edges to facilitate even filling (Figures 1B,C,E). The top plate is separated from a milled aluminum bottom plate via a 1/32″ sheet of silicone (Figures 1A,B,D), which serves as a gasket to seal the mold, maintaining pressure during injection, preventing leakage of the gelation mix, and preventing formation of air pockets. The pieces are held in place by clips placed on the long side of the mold (Figures 1A,C).

Fibrin gel fabrication was as described in Scruggs et al. (2025). Following curing at 37 °C, the process departs from that used in our prior work (Scruggs et al., 2025) as molds are opened and the gels hydrated in sterile PBS containing 2.5 mg/mL TXA and stored sterile at 4 °C (Figure 1E). At this point, the gels are blue in color due to the presence of trypan blue. Examination of gels using TEM showed that the gels have a fibrous structure similar to that observed previously (Gandhi et al., 2018; Scruggs et al., 2025; Gandhi et al., 2021) (Figure 1F), while SEM showed the surface to be relatively smooth with fibrils aligned parallel to the top surface plane interdigitated with crater-like voids across the surface (Gandhi et al., 2018; Scruggs et al., 2025; Gandhi et al., 2021) (Figure 1G). OCT measurements showed that gels were generally uniform in thickness averaging 182.9 ± 3.4 µm (mean ± SE, N = 6) with individual blanks varying in thickness by ± 11–20.3 µm with an average variance of 7.93% (Figures 1H,I).

For our purposes, the fibrin hydrogels must possess sufficient stiffness and elasticity to be handled and loaded in a surgical inserter instrument while being compliant enough not to damage surrounding tissues during placement. Young’s modulus for freshly fabricated gels was determined to be 0.053 ± 0.01 MPa, similar to our previously reported findings (Gandhi et al., 2018; Scruggs et al., 2025; Gandhi et al., 2021) and close to that of the retina, which is ∼0.02 MPa (Ferrara et al., 2021). Gels had sufficient stiffness and rigidity to utilize punching as a means to create smaller individual doses (Figures 2D–G).

A critical property of the gel is that it must be degradable, yet, not degrade during cell culture and storage. We have previously shown that gels produced using this or similar formulations are degradable (Gandhi et al., 2020; Gandhi et al., 2018; Scruggs et al., 2025; Gandhi et al., 2021); however, we have also shown that RPE cells grown on the fibrin will degrade the gel in the absence of aprotinin, a 6.5 kDa polypeptide protease inhibitor (Gandhi et al., 2019; Gandhi et al., 2018). Aprotinin is a part of the Tisseel kit, but we eliminated it because we found that it significantly impeded degradation of the gel and did not effectively wash out of the gel when used to stabilize it during cell culture (Gandhi et al., 2018; Scruggs et al., 2025). To address this, we substituted TXA a synthetic analog of lysine with a mass of 157 Da. Clinically, both are used to prevent bleeding; however, TXA is considered safer than aprotinin and is available over the counter in some countries. As shown in Figures 2I,J, culture of RPE cells on fibrin gels in media containing TXA results in a retention of the thickness of the gel.

For these studies, we used RPE cells derived from the iPSC line 22/1. RPE differentiated from 22/1 formed highly pigmented monolayers of cells (Figure 2) with a cobblestone appearance (Figures 2C,H). The cells exhibited integrin and MERTK-receptor dependent phagocytosis (Finnemann et al., 1997a) of photoreceptor outer segment fragments that is characteristic of RPE (Finnemann et al., 1997a) (Figures 3A–D) and secreted high levels of PEDF (Figure 3E). Using flow cytometry for CRALBP (Hill et al., 2024), the RPE were found to be 98.93% ± 0.74% pure with OCT3/4 expression <1.3 ± 1.6% (mean ± SD, n = 4) (Supplementary Figure 1). Residual iPSCs were assayed to <1 in 10,000 cells based on qPCR analysis of pluripotency markers ZSCAN10 and LIN28a (Hill et al., 2024) (Supplementary Figure 2).

Following seeding of RPE on the fibrin hydrogel, cells could be seen to adhere to the region of the top plate containing the gel. By 30 days after seeding, RPE cells could be seen as a pigmented layer of cells sitting atop the fibrin gel (Figures 2A–C,F). Inspection using a stereomicroscope demonstrated that the cells were uniformly distributed and pigmented across the gel (Figure 2B). Under higher magnification using a compound microscope, the cells were found to exhibit a cobblestone-like appearance (Figure 2C). While RPE cells often grew on the polycarbonate sides of the top plate (Figures 2A,B), they adhered poorly and often came off during feeding.

Between 30 and 60 days after seeding we used a custom oval punch (Figures 2D,E) to cut from 60–80 individual doses of ∼1.5 × 5.0 × 0.2 mm from each blank (Figures 2F–I). Each dose contains ∼30,000 cells. Visual inspection of individual doses demonstrated that the RPE were present on a single surface of the gel (Figures 2H–J) and did not appear to penetrate the gel (Figure 2I). It should be noted that handling the gels with forceps did cause damage to the RPE monolayer (left side of Figure 2H). As such, we avoid handling with forceps by using pipettes and pushing devices that did not damage the monolayer during manufacturing. Using OCT, we found that the thickness of the gel did not change during cell culture, and we could clearly distinguish the RPE from the gel based on differences in reflectivity (Figure 2J).

H&E staining (Figure 4A) and DIC imaging (Figures 4A,B,D,F) of paraffin embedded sections of RPE on fibrin hydrogels again showed that the thickness of the hydrogel appeared unaltered, that the RPE cells were present as a monolayer of pigmented cells on one surface of the gel, and that they did not penetrate the gel (Figure 4A). TEM found that the cells did not substantially alter the fibrous structure of the gel (Figure 4H) and that the iPSC-derived RPE had numerous basal infolds typical of RPE in situ (Figure 4I). Immunofluorescence staining showed that the cells retained expression and proper localization of select RPE phenotype markers; Tra-1-85 (CD147/EMMPRIN) (Figures 4B,C), Best1 (Figures 4D,E), and CRALBP (Figures 4F,G). The Tra-1-85 antibody recognizes human CD147/EMMPRIN, which is expressed uniquely by RPE cells in the eye (Finnemann et al., 1997b; Marmorstein et al., 1996; Marmorstein et al., 1998). Tra-1-85 staining of RPE-fibrin demonstrated the typical polarized distribution of CD147/EMMPRIN to the apical plasma membrane of RPE (Marmorstein et al., 1996; Marmorstein et al., 1998), and suggested that the cells had extensive microvilli (Figure 4C). Microvilli were confirmed by SEM (Figure 4I). Best1 exhibited basolateral polarity (Marmorstein et al., 2000). CRALBP is localized to intracellular compartments (Saari et al., 1984).

Individual doses were placed within the tip of a custom surgical inserter tool (Figures 5A,F). The tool is composed of two main pieces: a tip and a handle (Figure 5A). Both were designed to be for single use and were modified from previously described prototypes (Gandhi et al., 2020; Scruggs et al., 2025; Mano et al., 2022) as follows. The tip was designed to be consistent in diameter along its entire length to accommodate our findings on pressure maintenance during injection as described in Mano et al. (2022). By extending the tip width, a functional seal could be maintained during surgery that maintains intraocular pressure. The wire plunger used in early prototypes was replaced with a laser cut nitinol plunger coated with PTFE. Nitinol was chosen over a stiffer material like stainless steel as it allows some flexibility in the tip while still maintaining shape memory. The PTFE coating promotes a smooth motion of the plunger during insertion and provides protection against corrosion during storage in cell culture medium. This version of the tip was designed to be a storage container as well (Figures 5B,C). Once the dose is loaded in the tip, the tip is packaged in a sterile borosilicate glass R100 vial and filled with 100 mL of RPEM/B27/TXA (Figures 5B–E). The tip is held in place using a custom designed clip that is inserted in advance into the R100 vial (Figures 5C–E). The clip also prevents the dose from falling out during handling while still permitting contact with the cell culture medium in the vial (Figures 5D,E). Vials are crimp sealed and stored at 37 °C until use. Once packaged, the RPE survive and continue to produce PEDF for at least 7 weeks (Figure 5G). Assay of PEDF in the media found an average of 4.38 ± 2.70 µg (mean ± SD, N = 6) and 26.45 ± 2.89 µg (average ± SD, n = 3) of PEDF accumulated in the media at 2 and 7 weeks, respectively (Figure 5G).

For transplantation, the vial is opened and the culture medium decanted. The handle is then used to remove the tip and it is attached to a syringe and tubing (Figure 5F) that have been pre-filled with BSS or sterile water. Exerting pressure with the syringe causes the plunger to deploy the transplant (see Supplementary Video 1). Four domestic pigs underwent RPE debridement and RPE-fibrin transplant surgery as described in Section 4.7 and shown in Supplementary Video 1. The debridement zone, produced by gently scraping the RPE from Bruch’s membrane with a FINESSE® loop ranged from 7–13 mm² (Figures 6A,B). Following debridement, a 3.6 mm sclerotomy was made with an MVR blade. Argon laser was then applied to cauterize the choroid prior to entering the globe with a keratome blade. The transplant inserter was inserted through the sclerotomy, aligned with the retinotomy produced for debridement, and the transplant ejected into the subretinal space (Figures 6A,B) over the debridement zone. Following closure of the sclerotomy a fluid-air, and air-gas [Sulfur hexafluoride (SF6)] exchange was performed, and an intravitreal dexamethasone implant was placed in the remaining peripheral vitreous for immunosuppression.

Fundus exam including color fundus photos (Figures 6A,B,D–G), OCT (Figures 7A–C), and OCT-A (Figures 7D–K) were performed at 2 weeks, 1 month, and 2 months post-operatively (Table 1). By week 2, the gas bubble had dissipated in all pigs. Pigs 1 and 3 (Table 1) had vitreous hemorrhage at week 2 which prevented imaging but was cleared by 1 month. In pig 3 the vitreous hemorrhage obscured our view of the retina at 2 weeks. For Pigs 1, 2, and 4 (Table 1) the retina appeared flat over the debridement zone at week 2, though it remained elevated over the transplant site suggesting some residual fibrin scaffold remained. Where we could obtain cross sectional OCT through these areas however, it was noted that the elevated region was filled with hyper-reflective material suggesting inflammation rather than fibrin which is typically hypo-reflective on OCT. The fibrin scaffold in all four pigs was no longer visible and the retina was flattened by 1-month post-transplantation when examined using indirect ophthalmoscopy (Figure 6G) or OCT imaging (Figures 7A–C). Inflammation was observed in every pig receiving a transplant (Figure 8B). This is not unexpected, even with local immunosuppression (Ozurdex) since the RPE component of the transplant is xenogenic. OCT images outside the debridement zone showed normal retinal layers, and corresponding OCT-A flow overlay demonstrated normal flow signal throughout the CC. Regions in which the RPE were debrided exhibited GA and disciform scar in all four pigs after as little as 1 month with complete RPE and outer retinal atrophy (cRORA), pachyvessels, subretinal hyperreflective material (SRHRM), and inner choroidal thinning as well as other features that we have previously described in this model (Iezzi et al., 2024). Within the debridement zone, areas receiving RPE-fibrin transplants could be readily identified post-operatively by indirect fundoscopy as isolated islands of pigment within the cloudy white zone of debridement (Figures 6D–G). In transplant zones, there was no GA, disciform scarring, or cRORA observed (Figures 7A–C), though, similar to the debridement zone, there was SRHRM in all four pigs and pachyvessels in three (Table 1).

Analysis of en face OCT images was performed to compare the area covered by RPE-fibrin vs area debrided at 1-month post-op (Table 2). Since the debridement zone was created mechanically, there was some variability in the total area debrided. The area covered by transplanted RPE at 1 month averaged 4.41 ± 1.44 mm². The surface area of the RPE monolayer on an RPE-fibrin implant ranged between 6.31 and 6.42 mm². The difference in surface is consistent with some loss of cells due to contraction of the RPE monolayer post-implantation, as well as loss of cells due to immune rejection. Segmentation analysis of en face OCT-A images was performed to compare CC flow outside the debridement zone to zones of RPE debridement and debridement + RPE-fibrin (Figures 7F–L; Table 3). Consistent with our prior data (Iezzi et al., 2024), CC flow was significantly (p < 0.0001) reduced in the debridement zone compared to untreated regions outside of the debridement zone (Figure 7L). Zones receiving RPE-fibrin transplants also demonstrated significantly (p < 0.0001) less CC flow than regions outside the debridement zone (Figures 7F,G,L). However, RPE-fibrin transplant zones exhibited significantly (p < 0.0001) more CC flow than adjacent debridement zones (Figures 7D,G,H,J–L).

Examination of H&E-stained sections confirmed complete degradation of the fibrin scaffold in all four pigs. These sections also confirmed the loss of RPE in the debridement zone (Figure 8C) resulting in GA characterized by loss of photoreceptor outer segments. In addition, there was notable thinning of the outer nuclear layer (ONL), and immune cell infiltrates. In areas where transplanted RPE were present (Figure 8B), there was some thinning of photoreceptor outer segments, but, as observed by OCT, the ONL remained intact and overall retinal layering was preserved. In transplant zones, there were occasional cellular infiltrates that were absent from outside the debridement zone and debridement only zones. In general, the choroid appeared to be less affected (e.g., thicker) under transplants than in the debridement zone, confirming observations by OCT and OCT-A (Figure 7; Table 1). Immunofluorescence staining for human RPE marker CD147 (Figures 8D–G) confirmed the persistence of transplanted human RPE in the transplant zone even at 2 months post-transplantation (Figure 8E). It should be noted that while pig RPE do express CD147, the Tra-1-85 antibody is specific for human CD147 (Figure 8D). Staining similar to that observed in human retina (Figure 8F) and RPE-fibrin in vitro (Figure 8G) was observed in the zones receiving RPE-fibrin transplants (Figure 8E).

Gel fabrication was performed aseptically using a modification of our previous method (Scruggs et al., 2025). Fibrin hydrogels were produced using 4 mL Tisseel tissue glue kits (Baxter, cat# 1504518VP). The fibrinogen was resuspended in sodium citrate prepared from a clinical anti-coagulant solution (Fenwal Pharmaceuticals/NDC#-0942–9504–10) diluted to 0.01M with sterile water for injection (Gibco, cat# A1287301) rather than the included resuspension buffer which contains aprotinin. Thrombin was reconstituted with the thrombin solution from the same Tisseel Kit, and the vials incubated in a 37 °C water bath overnight. The following day, 0.6 mL of sterile tissue culture grade 0.4% trypan blue (Gibco, cat# 15250–061) was added to 2 mL of the resuspended fibrinogen and thrombin solutions. The fibrinogen solution was then drawn into an 11 mL syringe and the thrombin solution into a 1 mL syringe. The syringes were placed in a 11:1 ratio FibriJet Ratio Applicator Assembly. The gelation solution was then dispensed through a FibriJet Blending Connector with Mixer (Nordson Medical) and 18G cannula into a custom mold (Meddux, CO) (Figure 1). Molds were incubated at 37 °C for 3 h to allow gels to cure. Subsequently, molds were opened and top plates containing the polymerized gels were hydrated in sterile phosphate buffered saline (PBS) containing 2.5 mg/mL tranexamic acid (TXA). The resulting gel is ∼15.25 × 58.42 × 0.2 mm (0.7″ x 2.45″ x 0.008″) with a final concentration of >30 mg/mL of fibrin.

Gel thickness was measured using a Lumedica OQ Labscope optical coherence tomography (OCT) system at points of intersection of a 14 × 5 grid drawn on the top plate as described previously (Scruggs et al., 2025). Young’s modulus was determined at the Mayo Clinic Biomechanics Core Facility using a spherical indenter (radius = 0.25 mm) on a MicroTester G2 (CellScale, Waterloo, ON) as before (Scruggs et al., 2025).

Both scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed at the Mayo Clinic Microscopy and Cell Analysis Core as previously described (Gandhi et al., 2018; Scruggs et al., 2025; Gandhi et al., 2021) using samples fixed in Trumps fixative at 4 °C and embedded in plastic resin for TEM. 100 nm thin sections were viewed using a JEOL 1400 microscope (JEOL; Peabody, MA). SEM samples were viewed and imaged using a Hitachi S-4700 cold field emission scanning electron microscope.

The iPSC line 300-BIOTR-0022 clone 1 (22/1) was produced in compliance with cGMPs from fibroblasts obtained from a 25-year-old Caucasian female donor who met donor eligibility criteria (21 CFR 1271) (Marmorstein et al., 2023). 22/1 cells were maintained and differentiated to RPE at LAgen Laboratories (Rochester, MN) as previously described (Hill et al., 2024). All procedures were carried out aseptically by teams of two or more trained operators in Class 2A biosafety cabinets in an ISO 7 cleanroom. No antibiotics or antimycotics were used in cell culture. All reagents used were produced using GMPs with the exception of Accumax (Innovative Cell Technologies, cat# AM105) and were xenofree with the exception of mTeSR1 (Stem Cell Technologies, cat# 85850), which contains bovine serum albumin from BSE-free herds and is tested for bovine adventitious viruses. A Master Cell Bank (MCB) of iPSC line 300-BIOTR-0022 clone 1 was produced with 0.5 mL of cells cryopreserved in StemCell Banker (DMSO free, GMP grade, AMSBIO, cat# 13926) at 2 × 10⁶ cells/vial. The MCB tested sterile (USP<71>, Eurofins), mycoplasma free (PCR, LabCorp), and was free of adventitious viruses (Eurofins). For this study, two lots of RPE cells were produced each from an independent vial of cells. These were the same lots used in Hill et al. (2024). In brief, for RPE production a vial of iPSCs containing 2 × 10⁶ iPSCs was thawed and seeded into a T25 flask coated with Synthemax-2 SC (Corning, cat# 3535). Cells were fed daily with mTeSR1 and grown to confluence in a 37 °C 95% air/5% CO₂ incubator. Upon achieving confluence, media was changed to CTS-Knock-Out DMEM (GIBCO, cat#) containing 15% (v/v) CTS-Knock-Out SR xenofree medium (GIBCO, cat# 12618013), 1% (v/v) nonessential amino acids (GIBCO, cat# 11140050), 1% (v/v) GlutaMAX (GIBCO, cat# 35050061), and 0.1 mM 2-mercaptoethanol (GIBCO, cat# 21985023) and fed daily. After 49 days, cells were passaged using Accumax, filtered through a 40 µm nylon mesh (Corning, cat# 431750), re-suspended in RPEM/B27 [RPEM (LAgen Laboratories, see Supplementary Table 1) containing 2% CTS-B27 Xenofree supplement (GIBCO, cat# A1486701)] and seeded at 1 × 10⁷ cells in Synthemax-2 SC coated T25 flasks. Cells were fed every other day for 30 days and then passaged again, as described above, prior to being seeded on fibrin gels at 1 × 10⁷ cells/gel blank in RPEM/B27 containing 2.5 mg/mL tranexamic acid [(TXA), Provepharm, NDC# 81284–611–00] (RPEM/B27/TXA). Prior to seeding with iPSC-RPE, gel blanks were washed three times for 20 min in RPEM/B27/TXA. The gel was placed in a 95% air/5% CO₂ incubator at 37 °C and cells were fed with RPEM/B27/TXA every 2 days for 30–60 days after which individual doses were prepared and packaged as indicated below.

A transplantation device that also served for safe storage of the RPE/fibrin gel was developed with assistance from Meddux Corp (CO) based on the prototypes described in Gandhi et al. (2020) and Mano et al. (2022). The current device consists of a disposable handle and tip assembly (Figure 5). The reusable handle has a pneumatic actuator, luer-lok connector, and threads for the disposable tip assembly. The disposable tip assembly consists of a spring-guided pin attached to a polytetrafluoroethylene (PTFE) coated nitinol plunger that is housed within a plastic tube shaped to fit the nitinol plunger. The RPE/fibrin hydrogel transplant is punched from the blank using a custom machined stainless steel punch after 30–60 days of culture. The resultant 1.5 × 5.0 × 0.2 mm oval “doses” were then loaded into tip. Following visual inspection for defects in the cell monolayer and to insure orientation of the monolayer, tips were placed in a custom clip in an R100 vial filled with 100 mL of RPEM/B27/TXA and crimp sealed. The packaged transplants were stored at 37 °C until used. During surgical transplantation the R100 vial was opened using a de-crimping tool, the media decanted, and the inserter handle screwed onto the tip to remove it from the vial. Once the handle and tip were ready, the handle was connected via a Luer fitting to a plastic tube and 6cc syringe filled with balanced salt solution. To deploy the transplant from the housing, pressure is applied via syringe to the pneumatic actuator causing the transplant to be expelled.

Flow cytometry was performed as described previously (Hill et al., 2024) for CRALBP with mouse anti-CRALBP monoclonal antibody B2 (NOVUS, Cat# NB100-74392, RRID: AB_1048601) followed by a PE coupled Goat anti-Mouse IgG1 secondary antibody (Invitrogen, cat# P-21129, RRID: AB_1500811). Flow cytometry for OCT3/4 was performed as described in Hill et al. (2024) using Alexa Flour 488 conjugated mouse anti-Oct3/4 (BD Pharmingen, cat# 560253, RRID: AB_1645304). Data were acquired using a BD FACS Aria II (BD Biosciences) and analyzed using Flowjo Software (Tree Star).

QPCR was performed using 10 ng of cDNA and TaqMan™ Gene Expression Assays for ZSCAN10 (Hs00262301_m1), LIN28A (Hs04189307_g1), and β-actin (Hs01060665_g1) as described previously (Hill et al., 2024).

Pigment epithelium derived factor (PEDF) was assayed by ELISA, as previously described (Johnson et al., 2015; Johnson et al., 2013) using conditioned medium collected from P0 cells at 49 days and for P1, P2, and RPE-Fibrin at 30 days post-plating. The phagocytosis function of iPSC RPE was determined as before (Muller et al., 2018). In brief, photoreceptor outer segment fragments (POS) were prepared from fresh pig eyes and stored as frozen stock at −80 °C (Parinot et al., 2014). Thawed POS were covalently labeled with Texas Red by incubation with Texas Red™-X Succinimidyl Ester (Thermofisher, cat# T6134) at 30 μg/mL in 0.1M Na-bicarbonate buffer pH 9.5 for 1 h on the day of the experiment. iPSC RPE on glass coverslips were serum starved for 1 h prior to incubation for 5 h with labeled POS at a concentration of ∼10 particles per cell. POS were fed in DMEM alone, or in DMEM supplemented with purified human MFG-E8 (Biotechne cat# 2767-MF) and Protein-S (Biotechne, cat# 9489-PS-100), at 2 μg/mL each in the presence of 1% PBS (as solvent control) or of 1% cilengitide (MilliporeSigma, cat# 188968–51–6) stock solution (100 μM in PBS). Cells were washed three times with PBS, fixed with 4% paraformaldehyde, and counterstained with 4′,6-Diamidino-2-Phenylindole (DAPI) before mounting on microscope slides. Phagocytosed POS were quantified by flatbed fluorescence scanning and visualized on a Leica TSP8 confocal microscopy system, as described previously (Muller et al., 2018). Four independent phagocytosis assays with triplicate samples each were performed and analyzed.

All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Mayo Clinic and conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and National Institutes of Health guidelines. Female domestic pigs (Sus scrofa domesticus) at 2 to 3-month of age and weighing between 20 and 35 kg were used for this study. For all animals the right eyes (OD) were designated as the experimental/operative eyes. RPE-fibrin transplantation was performed by two different surgeons (RI, pigs 1-3, BAS pig 4; see Table 1), as described previously (Gandhi et al., 2020) following RPE debridement as before (Iezzi et al., 2024) (see Supplementary Video 1). Two different blanks were used each with a distinct lot of iPSC-RPE as indicated in Table 1. RPE-Fibrin was surgically implanted within 18 days of packaging. All operated eyes received a dexamethasone implant (Ozurdex®, Allergan, NDC No. 0023–3348–07) for immunosuppression which was placed in the remaining peripheral vitreous at the end of the procedure.

Pigs were anesthetized using isoflurane, as before (Gandhi et al., 2020; Scruggs et al., 2025; Iezzi et al., 2024) for each post-operative examination. Eye drops were instilled for dilation and topical anesthesia as above. Color fundus photos, OCT, and OCT-Angiography (OCT-A) were performed at each exam. Fundus photographs were obtained using a custom-made video indirect ophthalmoscope. Images were processed using Photoshop (Adobe, San Jose, CA). OCT, OCT-A, and infrared SLO were performed using the Optovue Avanti OCT Angiovue System (Visionix; North Lombard, IL).

Quantification of choriocapillaris flow was performed using 6 × 6 mm OCT-A scans. For each pig, five regions outside the RPE debridement zone (native RPE), five regions within the RPE debridement zone, and five regions within the transplant region (RPE debridement + RPE-fibrin) were randomly selected based on high-quality OCT-A image acquisition. Each region was analyzed using Optovue’s flow area analysis software. To standardize quantification across pigs, we used a predefined 0.5 mm diameter measurement circle, corresponding to an approximate area of 0.2 mm². Flow area within each circle was quantified as the fraction of pixels exhibiting flow signal, as determined by the device’s built-in flow detection algorithm within the segmented choriocapillaris slab.

Pigs were euthanized at 1 or 2 months post-op (Table 1) by rapid intravenous injection of a pentobarbital solution [FATAL-PLUS, Vortech (NDC No. 0298–9373–68); 1 mL/per 10 lbs of body weight]. Eyes were enucleated, fixed in Davidson’s fixative, and processed into paraffin, as previously described (Scruggs et al., 2025). iPSC-derived RPE on fibrin gels were fixed in neutral buffered formalin prior to embedding in paraffin. 5 μm sections were cut and either stained with H&E or processed for immunofluorescence as described below. H&E stained sections were scanned using a Leica Aperio® AT2 microscope slide scanner and viewed and images captured using Aperio Imagescope software. Human Eyes were fixed and processed as described in Marmorstein et al. (2002).

RPE monolayers on fibrin hydrogels were fixed in neutral buffered formalin for >24hrs, then processed for paraffin histology. Immunofluorescence was performed, as described previously (Scruggs et al., 2025; Hill et al., 2024), using rabbit polyclonal antibody Pab125 to detect Best1 (Marmorstein et al., 2000), CD147/EMMPRIN using mouse monoclonal antibody Tra-1-85 (R&D Systems, cat# MAB3195, RRID: AB_2066681), or CRALBP using mouse monoclonal antibody B2 (Novus, cat# NB100-74392, RRID: AB_1048601). Nuclei were counterstained using 4′,6-diamidino-2-phenylindole (DAPI). Immunofluorescence and transmitted light images were obtained on a Nikon E600 microscope (Nikon Instruments, Melville, New York, USA) using a CCD camera and Nikon Elements Software.

Data are presented as mean ± standard deviation (SD) or standard error (SE) as indicated. Statistical significance was determined using the TTEST function in Microsoft Excel, except for phagocytosis assays where statistical significance was determined using one-way ANOVA and Tukey post hoc testing (GraphPad Prism).