Development of stromal corneal grafts using a novel decellularization method with sodium cocoyl glutamate on GGTA1/CMAH/β4GalNT2 knock-out porcine corneas

Introduction

Background

The human cornea is a transparent tissue located at the front of the eye. It is crucial for vision as it must effectively transmit visible light (1,2). Transparency of the cornea can be compromised by various diseases (3). Corneal damage caused by infection or trauma can result in severe corneal opacity, which impairs the cornea’s ability to effectively transmit visible light (4). In addition to diseases that cause corneal opacity, there are conditions where the cornea protrudes forward and loses its function as a uniform refractive surface, with keratoconus being a representative example (5,6).

The cornea is composed of three main layers: the corneal epithelium outer layer, which is in contact with tears on the surface; the corneal stroma middle layer, which constitutes most of the corneal thickness; and the corneal endothelium inner layer, an important cell layer on the innermost side of the cornea that maintains its transparency by regulating corneal hydration status (2,4).

For cases with corneal edema and opacity caused by abnormalities in corneal endothelial cells, endothelial keratoplasty has recently become widely performed (7,8). This procedure involves replacing the damaged corneal endothelial cells with healthy donor endothelial cells (8). When the fluid pumping function of endothelial cells is restored, corneal edema decreases and corneal opacity significantly improves. However, if the corneal pathology is located in the stroma rather than the endothelial layer, the transparency of the cornea cannot be restored if the stromal part is not replaced with donor cornea (9). Since the stromal part also requires donor cornea, in regions with a shortage of corneal donors, many people are left waiting for corneal transplants (10,11).

Rationale and knowledge gap

Chronic shortage of human donor corneas has sparked significant interest in xenografts, particularly using pig corneas (12-16). This field has seen steady progress, with a recent breakthrough by a Chinese research team achieving clinical commercialization by decellularizing pig corneal stroma, yielding satisfactory clinical results (17-19). Pigs are favored due to their genetic similarity to humans, ease of breeding, and corneal size similarity to human corneas (20). However, xenografts in ophthalmology face challenges such as immune rejection, zoonotic disease transmission, and ethical concerns (13,21). Physiological differences between human and animal tissues can impact graft success. Ensuring the viability and functionality of xenografts over time is challenging, but advancements in genetic engineering and decellularization techniques offer promise (13,14,22-24). Genetically engineered pigs with reduced antigenicity and decellularized porcine corneal stroma have shown potential in minimizing immune rejection and improving clinical outcomes (14,22,23).

Objective

In this study, GGTA1/CMAH/β4GalNT2 knockout pigs were created. Knocking out the GGTA1, CMAH, and β4GalNT2 genes is crucial for reducing immune rejection in xenotransplantation (25-27). These genes produce carbohydrate antigens such as galactose-alpha-1,3-galactose (α-Gal), N-glycolylneuraminic acid (Neu5Gc), and Sd antigens that the human immune system recognizes as foreign. Eliminating these antigens can reduce binding of human antibodies to pig cells, preventing hyperacute rejection and improving survival of transplanted tissues or organs. Corneal stroma harvested from these pigs can be decellularized to create corneal grafts. This study aimed to introduce a new decellularization method using sodium cocoyl glutamate (SCG) and compare it with the existing method using sodium N-lauroyl glutamate (SLG) (28). To evaluate the safety and efficacy of a novel decellularization method using SCG across diverse models, corneas harvested from wild type (WT) and GGTA1/CMAH/β4GalNT2 knock-out [triple knockout (TKO)] pigs were transplanted into rabbits. We present this article in accordance with the ARRIVE reporting checklist (available at https://atm.amegroups.com/article/view/10.21037/atm-25-3/rc).

Methods

Animals

Animals were treated in compliance with national guidelines for the care and use of animals. The experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the Biotechnology Research Institute of MGENSolutions Co., Ltd., Republic of Korea (IACUC reference number: 2019-1). A protocol was prepared before the study without registration. All surgical procedures were conducted under general anesthesia. Efforts were made to minimize any potential suffering of animals. All animals were raised under conventional housing conditions. Rabbits were purchased from the approved local vendor (Orient Bio Inc., Seongnam, Republic of Korea).

Production of GGTA1/CMAH/β4GalNT2 knockout pigs

Using an online CRISPR design tool (http://chopchop.cbu.uib.no/), single guide RNA (sgRNAs) that could recognize porcine GGTA1, CMAH, and β4GalNT2 genes were designed. These oligomers were synthesized and introduced into the pCas9-H2Kk-PM vector. CRISPR vectors for editing porcine GGTA1, CMAH, and β4GalNT2 genes were introduced into the porcine primary fibroblasts by electroporation method using Nucleofector^TM (LONZA, Basel, Switzerland). After 48 h, transfected cells were sorted for H-2Kk using MACSelect^TM Kk System (Miltenyi Biotec Inc., USA) to enrich genetically modified cells. Sorted cells were cultured in 100 mm culture dishes. A portion of cultured cells was used for genotyping. Genomic DNAs were extracted from cultured cells using a genomic DNA extraction kit (iNtRon Biotechnology, Seongnam, Republic of Korea) following the manufacturer’s instructions. Extracted genomic DNA was used as a template for polymerase chain reaction (PCR). PCR amplicons were used for T7 endonuclease I (T7E1) assay for checking mutation. Somatic cell nuclear transfer (SCNT) was then performed as described previously (29). Briefly, pig oocytes were obtained from ovaries collected from a local abattoir. After 44 h of maturation, matured oocytes were enucleated and then a single donor cell was injected into the perivitelline space of enucleated oocytes. Oocyte cytoplasm-cell complexes were then fused and activated by electric pulse. Reconstructed embryos were surgically transferred into the oviduct of a surrogate. After approximately 114 days, cloned piglets were naturally delivered. A tail biopsy was performed for each piglet for genomic DNA extraction and genotyping. To check genetic modifications of each piglet, PCR and T7E1 assay were conducted as described above.

The presence of α-Gal, Neu5Gc, and β4Gal antigens was analyzed through flow cytometry using peripheral blood mononuclear cells (PBMCs). Whole blood samples were obtained from each piglet, and PBMCs were subsequently isolated using Ficoll-Paque^TM PLUS (GE Healthcare, IL, USA). To detect specific antigens, the isolated PBMCs were labeled with fluorescein isothiocyanate (FITC)-conjugated isolectin B4 (1:100, Enzo Life Science, NY, USA) for α-Gal, anti-Neu5Gc antibody (1:200, BioLegend, CA, USA), and Alexa Fluor^® 647-conjugated AffiniPure F(ab')2 Fragment Donkey anti-chicken IgY (1:100, Jackson ImmunoResearch, PA, USA) for HD antigens. Additionally, FITC Dolichos biflorus agglutinin (DBA) lectin antibody (1:200, BioLegend, CA, USA) was used to stain β4Gal antigens. Flow cytometric analysis was performed using BD Accuri^TM C6 Plus (BD Bioscience, CA, USA). Immunohistochemistry was conducted using corneal tissues from a wild-type pig and generated knockout piglets. Corneal tissues were fixed in 4% paraformaldehyde (PFA), embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). These specimens were treated with a primary antibody (anti-pig antibody) followed by a secondary antibody (FITC or Alexa 488 conjugated anti-immunoglobulins). Nuclear staining was performed with DAPI. Finally, specimens were observed under a laser scanning confocal microscope.

Decellularization process

The decellularization method was carried out by adapting techniques introduced in previous studies (28,30). Fresh porcine eyes were obtained within 2 h post-mortem and washed with phosphate-buffered saline (PBS) containing 1 mg/mL tobramycin for the cleaning. About 15-mm diameter sized corneoscleral button was gently dissected from the eyeball using surgical blade and scissors. The central corneas were punched using 8-mm diameter sized Barron vacuum donor cornea punch (Catalog No. K20-2108, Katena product Inc., Corza ophthalmology, NJ, USA). Before proceeding to the decellularization process, porcine corneas were washed with 10 mL of PBS with 5% penicillin-streptomycin 3 times. Dissected round corneas were then randomly divided into groups and decellularized with the following treatments. Native corneas meant fresh native porcine corneas without any treatment. To compare decellularization methods, the porcine cornea was separately soaked with either 200 U/mL supernuclease (SN) (Sino Biological Inc., Beijing, China), 0.5% SLG (Sigma-Aldrich, St. Louis, MO, USA), 0.1% SCG, 0.5% SCG with 200 U/mL SN, or 1% SCG with 200 U/mL SN for 2 h at room temperature under a shaking condition (100 rpm). The decellularized corneas were then rinsed 10 times (30 min each time) with 20 mL of PBS, followed by a final rinsing with 20 mL of normal saline (0.9% NaCl). All treatments were performed under shaking (100 rpm). Finally, decellularized porcine corneas were dehydrated in glycerol.

Optical transparency measurement

Optical properties of corneas were assessed using a ultraviolet-visible (UV-Vis) spectrophotometer. Briefly, 6-mm diameter central corneal pieces were trephined and immersed in glycerol overnight. Light transmittance was measured every 100 nm wavelength ranging from 300 to 900 nm wavelengths using a UV-Vis recording spectrophotometer (SpectraMax M2, Molecular Devices, MD, USA). Transmittance of each sample was corrected with glycerol as a blank medium. Data were analyzed as mean percentage of transmittance. All characteristics were tested with native porcine corneal stroma as controls.

Tensile strength measurement

The tissue was immersed in a medium containing 15% dextran for 24 h to achieve dehydration similar to the situation in vivo. Central tissue samples were cut with a 15-mm diameter trephine and central part of the tissue was dissected to make 15 mm × 2 mm size strips. The tensile strength was measured by the Korean Confirmatory Laboratories, an accredited testing institution recognized by the Korean government.

DNA content measurement

Porcine corneas were dried and their weights were measured. Corneas were homogenized and DNAs were extracted using a genomic DNA extraction kit (iNtRon Biotechnology) following the manufacturers’ instructions. DNA concentration was quantified photometrically at a wavelength of 260 nm using a NanoDrop OneC Microvolume UV-Vis spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA).

Glycosaminoglycan (GAG) content determination

The GAG content of cornea was determined using a 1,9-dimethyl methylene blue (DMMB) assay (280560-N, Proteoglycan Detection Kit, Amsbio, Cambridge, Massachusetts, USA) according to the manufacturer’s protocol. The central 6 mm of the cornea was trephined, homogenized, and digested with papain at 60 ℃ for 1 h, followed by addition of acetic acid and Tris-hydrochloric acid. Digested samples were then added to DMMB and absorbance was measured at 515 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Collagen content measurement

For measuring total collagen content, protein fraction of the cornea was processed with an AllPrep DNA/RNA/Protein Mini Kit (Qiagen, Germantown, Maryland, USA). Collagen quantification was performed using a colorimetric assay (Sircol Collagen Assay, Biocolor, UK) according to the manufacturer’s instructions. Samples were measured at 550 nm using a plate reader (Molecular Devices, Sunnyvale, CA, USA) and collagen content was determined using a standard curve provided by the Sircol assay.

Preparation of cornea stromal graft for animal experiment

A fresh porcine cornea was mounted in an artificial chamber. The cover was tightened to create a watertight environment. Then pre-connected saline was infused at a constant pressure to maintain corneal pressure and a keratome was used to perform lamellar dissection to obtain a stromal graft with a thickness of approximately 200 micrometers (Figure 1A). After obtaining lamellar graft from the mid stroma, a centered round trephination was performed using 4-mm diameter of corneal punch. The round stromal graft was then processed with decellularization using 0.5% SCG combined with SN. After sterilization using UV radiation, the stromal graft was air dried and packaged in sterile condition for use in animal experiments (Figure 1B).

Figure 1 Preparation of lamellar cornea graft and insertion into rabbit cornea. (A) A fresh porcine cornea is mounted in an artificial chamber (a) and the cover is tightened to create a watertight environment (b). Then pre-connected saline is infused at a constant pressure to maintain corneal pressure (c) and a keratome is used to perform lamellar dissection to obtain a stromal graft with a thickness of approximately 200 micrometers (d). (B) The stromal graft is decellularized and dried to create a 4-mm diameter circular graft in dry form. (C) After general anesthesia, a 30-gauge needle is used to inject saline into the corneal stroma of the rabbit to induce corneal edema, facilitating lamellar dissection. A stromal pocket was then created using a corneal dissector and the prepared stromal graft was inserted into the pocket, followed by corneal suturing. (D) The anterior segment photograph taken immediately after surgery shows corneal edema intentionally made for stromal pocket formation, the circular graft inserted into the stromal pocket, and corneal sutures.

Animal experiment and evaluation

Male, specific pathogen-free (SPF), New Zealand White rabbits (n=20, 2.0–2.5 kg) were purchased from Orient Bio Inc. (Seongnam, Republic of Korea). Rabbits were provided with adequate food, water, ventilation, temperature (23 ℃, a 12 h/12 h daylight cycle) and humidity (45%) throughout the experiment.

Animals were assigned to treatment groups completely at random, without considering any other variables. The rabbits were divided into four groups (n=5, each group) and each group received a corneal transplant: WT, WT decellularized, TKO, or TKO decellularized. The sample size for an animal study was calculated to compare performances of two corneal grafts based on outcomes measured on a scale of 1 to 4. When the expected difference in transparency between the two grafts was set at 1, with a standard deviation of 0.5, a significance level of 0.05, and a power of 0.8, at least 4 animals per group were needed. Therefore, we included 5 rabbits in each group.

After general anesthesia using tiletamine and zolazepam (Zoletil^TM, Virbac, Republic of Korea), a rabbit’s eye was exposed using a speculum. A 30-gauge needle was carefully inserted at the limbus and normal saline was slowly injected into the corneal stroma to induce marked corneal edema. This process facilitated corneal lamellar dissection while minimizing the risk of full-thickness penetration by the dissecting blade. A 4-mm partial corneal incision (main incision) was made near the limbus, followed by blunt lamellar dissection. An 8-mm diameter corneal lamellar dissection created a stromal pocket large enough to snugly insert the corneal stromal graft. After checking graft position inside the pocket, the main incision was sutured and closed with one or two 10-0 nylon to prevent the graft-wound extrusion (Figure 1C,1D).

Rabbits were evaluated immediately after the surgery. They were also evaluated on day 1, week 1, week 2, and week 4 under a microscope. An anterior segment photograph was obtained at each time point. To evaluate corneal neovascularization, a grading system was used to determine the severity (grade 1, a clear cornea with peripheral vascularization of less than 2 mm; grade 2, peripheral vascularization of more than 2 mm, but not extending into the central cornea; grade 3, vascularization in the central cornea; and grade 4, vascularization in the central cornea accompanied by fibrosis) (31). The severity of corneal opacification was graded from 1 to 4 (1, a clear cornea with clearly visible iris details; 2, partial obscuration of the iris details; 3, poor visibility of iris details with a barely visible pupil margin; and 4, completely obscured iris and pupil details) (32). Although the evaluation procedures themselves did not cause pain to the animals in this experiment, efforts were made to ensure the welfare of the animals throughout the entire experimental period. The treatments were carried out by skilled experimenters to minimize the stress on the animals and any confounders such as the order of treatments and measurements as much as possible.

After completing the 4-week observation period, rabbits were euthanized by intravenous injection of potassium chloride (KCl, 2 mmol/kg) under deep anesthesia using overdose (50 mg/kg) of tiletamine and zolazepam (Zoletil^TM, Virbac, Republic of Korea) intramuscular injection in accordance with the guidelines of the institutional ethics committee. After the sacrifice of the animals, their eyeballs were harvested. Corneal optical coherence tomography (OCT) images were obtained using optical coherence tomography (Cirrus, ZEISS, Germany). Eyeballs were then bisected at the equator and the anterior portion was prepared for tissue slides.

Histology and immunohistochemistry

Tissue samples were preserved in 10% formalin, embedded in paraffin, and sectioned to a thickness of 4 µm. Histological analysis included H&E staining, along with immunostaining procedures. Specifically, paraffin-embedded tissues were sliced into 5-µm sections and subjected to permeabilization using 0.3% Triton X-100 in PBS for 10 minutes. The sections were then blocked with 10% normal goat serum (NGS) and incubated overnight at 4 ℃ with primary antibodies diluted in 1% NGS in PBS. Detection was carried out using fluorescein isothiocyanate-conjugated or rhodamine-conjugated anti-mouse or anti-rabbit immunoglobulins (Life Technologies, Carlsbad, CA, USA). For nuclear staining, DAPI (Vectashield antifade mounting medium with DAPI; Vector, Burlingame, CA, USA) was applied. Immunolabeled cryosections were observed under a laser scanning confocal microscope. As negative controls, the primary antibodies were substituted with equimolar amounts of an unrelated isotypic primary antibody from the same species.

Statistical analysis

Data are presented as mean ± standard error. Statistical significance was determined using a one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. In this study, statistical significance was indicated by asterisks using New England Journal of Medicine formatting for P values. Calculations were completed with GraphPad Prism Ver. 7.01 (GraphPad Software, Inc., La Jolla, CA, USA).

Results

Verification TKO pig

TKO pigs were produced by targeted deletion of three specific genes: GGTA1, CMAH, and β4GalNT2. PCR and T7E1 analysis were utilized to measure expression levels of these genes, revealing significant reductions of these genes in TKO pigs than in wild-type (WT) pigs (Figure 2A). These reductions indicated successful knock-out of these genes. DNA Sanger sequencing analysis confirmed the findings (Figure 2B). Additionally, flow cytometry analysis of PBMCs from TKO pigs showed marked decreases of expression levels of GGTA1, CMAH, and β4GalNT2, further confirming successful gene knock-out at the cellular level (Figure 2C). These findings collectively demonstrated the effectiveness of the gene editing process in producing TKO pigs.

Figure 2 Production of TKO pigs. (A) PCR and T7E1 analysis demonstrate that target regions of GGTA1, CMAH, and β4GalNT2 genes are significantly deleted in TKO pigs compared to WT pigs, indicating successful gene knock-out. The gel images were un-cropped whole gel images. The inserted graph compared the quantification of band intensity of T7E1 analysis. WT: wild type pig; Donor: triple knockout cells produced as cell donors; L1: a cloned animal produced from single donor cell by somatic cell nuclear transfer method. (B) The result of DNA sanger sequencing analysis of WT and L1. (C) Flow cytometry of peripheral blood mononuclear cells from TKO pigs shows markedly decreased expression of GGTA1, CMAH, and β4GalNT2, further confirming their knock-out at the cellular level. (D) Immunostaining of corneal tissue reveals that GGTA1, CMAH and β4GalNT2, which are present in WT corneas, are absent in TKO corneas, highlighting the effectiveness of the gene knock-out in corneal tissues. Scale bar indicates 100 µm. APC, allophycocyanin; FITC, fluorescein isothiocyanate; PCR, polymerase chain reaction; T7E1, T7 endonuclease 1; TKO, triple knockout; WT, wild-type.

To further validate gene knock-out, immunostaining of corneal tissues was performed. GGTA1 and β4GalNT2 proteins were present in WT corneas but absent in TKO corneas, confirming successful knock-out of these genes specifically in corneal tissues (Figure 2D). Such knock-out is crucial for applications in corneal transplantation and tissue engineering. The combination of PCR, flow cytometry, and immunostaining provided a comprehensive confirmation of gene knock-out. It ensures that TKO pigs are suitable for further research and potential clinical applications.

No gross abnormalities were observed in eyeballs of TKO pigs, indicating that the gene editing process did not adversely affect the overall structure of the eye. When corneal thicknesses were compared between TKO pigs and WT pigs, it was found that corneas of TKO pigs were significantly thinner than those of 2-month-old WT pigs (Figure 3A). Despite the observed difference in corneal thickness, the tensile strength of the cornea did not show a significant difference between 2-month-old TKO pigs and WT pigs (Figure 3B). This finding indicated that, although the corneal thickness was reduced in TKO pigs. The mechanical integrity and resistance to deformation of the cornea remained comparable to those of WT pigs. The average central corneal power was measured to be 45.38 diopters for 2-month-old TKO pigs and 37.90 diopters for WT pigs.

Figure 3 Corneal thickness and tensile strength were compared between TKO pigs and WT pigs. (A) Corneal thickness of TKO pigs was significantly thinner than those of 2-month-old WT pigs. (B) There was no significant difference in tensile strength of cornea between 2-month-old TKO pigs and WT pigs. ***, P<0.001. d, days; mo, months; ns, not significant; TKO, triple knockout; wk, weeks; WT, wild-type.

Decellularization of cornea

Corneal transparency of 2-month-old TKO pig corneas was evaluated after decellularization using different methods involving SLG and SCG (Figure 4A). Results showed that the decellularization method combining 0.1% and 0.5% SCG with SN achieved corneal transparency similar to that with a method using 0.5% SLG with SN. However, when 1.0% SCG was used with SN, corneal transparency showed a noticeable decrease. In addition, the combination of SN with either SLG or SCG was more effective in reducing residual DNA than using SLG or SCG alone. Among SCG concentrations tested, 0.5% SCG was more effective in reducing residual DNA than 0.1% SCG (Figure 4B). Furthermore, the amounts of glycosaminoglycan and collagen in corneal tissues showed no significant differences across all tested decellularization methods compared to pre-decellularization levels, suggesting that these methods did not adversely affect structural components of the cornea (Figure 4C,4D). Histologic evaluation revealed no significant change of collagen lamellar structures after decellularization (Figure 4E).

Figure 4 Cornea evaluation after decellularization. (A) Comparison of corneal transparency after decellularization using SLG and SCG. The experiment was conducted using corneas of 2-month-old triple knock-out pigs. Compared to the method using 0.5% SLG and SN, the decellularization method combining 0.1% and 0.5% SCG with SN achieved similar corneal transparency. However, using 1.0% SCG with SN resulted in a noticeable decrease in transparency. (B) The total amount of residual DNA in corneal tissue after decellularization was compared. The combination of SN with either SLG or SCG effectively reduced residual DNA compared to using SLG or SCG alone. Additionally, 0.5% SCG was more effective in reducing residual DNA than 0.1% SCG. (C,D) The amounts of glycosaminoglycan and collagen showed no significant differences among all tested decellularization methods compared to pre-decellularization levels. (E) Hematoxylin and eosin staining of native and decellularized cornea shows intact corneal structures. Black scale bar: 50 µm. *, P<0.05; **, P<0.01; ***, P<0.001. GAG, glycosaminoglycan; ns, not significant; SCG, sodium cocoyl glutamate; SLG, sodium N-lauroyl glutamate; SN, supernuclease.

This study found that using 0.5% SCG with SN for decellularization did not significantly affect tensile strengths of corneas from 2-month-old TKO pigs (Figure 5). This indicates that this method can preserve the mechanical integrity of the corneal tissue, making it suitable for corneal transplantation and tissue engineering.

Figure 5 The decellularization method combining 0.5% SCG and SN did not significantly affect tensile strength of cornea. The experiment was conducted using corneas of 2-month-old triple knockout pigs. SCG, sodium cocoyl glutamate; SN, supernuclease.

In vivo evaluation

Severe neovascularization developed at the corneal limbus by the first week in rabbits transplanted with WT or decellularized WT corneas. By the second and fourth weeks, severe corneal opacity and inflammation around the graft or partial graft melting were observed. In contrast, rabbits transplanted with TKO and decellularized TKO corneas showed clear grafts without significant inflammation or neovascularization (Figure 6A).

Figure 6 In vivo evaluation of cornea graft. (A) Representative anterior segment photographs of rabbits after corneal stromal transplantation. Edema was commonly observed on the first day post-surgery. Severe neovascularization developed at the corneal limbus by the first week in rabbits transplanted with WT or decellularized WT corneas. By the second and fourth weeks, severe corneal opacity and inflammation around the graft or partial graft melting were observed. In contrast, rabbits with TKO and decellularized TKO corneas showed clear grafts without significant inflammation or neovascularization. (B-E) Anterior segment photograph and OCT image of a rabbit cornea at four weeks after transplantation with a WT (B), decellularized WT (C), TKO (D), and decellularized TKO (E) cornea. Severe neovascularization and opacity were observed, with OCT showing significant graft swelling and increased signal intensity both in WT and decellularized WT corneas. In contrast, TKO and decellularized TKO cornea grafts maintained transparency and minimal vascularization after four weeks. Histology (H&E staining; scale bar: 100 µm) revealed residual nuclear structures of WT and TKO graft (red arrows). Suspected infiltrating inflammatory cells (red arrowhead) were found in decellularized WT graft. Immunostaining confirmed strong CD3-positive cell infiltration (white arrows) in WT graft and significant CD3- or CD5-positive inflammatory cells in the decellularized WT graft (yellow arrows). Yellow squares were magnified to clearly demonstrate inflammatory cells in panel C. However, no inflammatory cells infiltration was observed both in TKO and decellularized TKO grafts. Scale bar: 100 µm. OCT, optical coherence tomography; SCG, sodium cocoyl glutamate; SN, supernuclease; TKO, triple knockout; WT, wild-type.

Four weeks post-transplantation, anterior segment photographs and OCT images of corneas in rabbits transplanted with decellularized WT corneas revealed severe neovascularization and opacity, with OCT showing significant graft swelling and increased signal intensity (Figure 6B,6C). Histology revealed infiltrating inflammatory cells in the decellularized graft, confirmed by immunostaining for CD3 and CD5. Conversely, decellularized TKO cornea grafts maintained transparency and minimal vascularization after four weeks. OCT showed a high signal at the graft boundary, although the internal graft had a homogenous signal intensity similar to the recipient cornea. Histology showed no inflammatory cells infiltrating the graft. Immunostaining for CD3 and CD5 showed no T cells infiltrated (Figure 6D,6E). Grading-based quantitative analysis also revealed these clinical differences (Figure 7). Four weeks after corneal stromal transplantation, severe neovascularization and corneal opacity were observed in rabbits transplanted with WT or decellularized WT corneas, while only mild neovascularization and opacity were seen in rabbits transplanted with TKO or decellularized TKO corneas, showing no significant differences between TKO and decellularized TKO groups. These results confirmed significant disparities in corneal neovascularization and opacity between groups, highlighting superior performances of TKO and decellularized TKO corneas to WT and decellularized WT corneas.

Figure 7 Comparison of corneal neovascularization and corneal opacity between groups. Four weeks after corneal stromal transplantation surgery, corneal neovascularization and corneal opacity were compared between groups in rabbit corneas. In rabbits transplanted with WT and decellularized WT corneas, severe neovascularization (A) and corneal opacity (B) were observed. However, in rabbits transplanted with TKO or decellularized TKO corneas, only mild neovascularization and corneal opacity were observed. No significant differences were found between TKO and decellularized TKO groups (n=5 for each group; ***, P<0.001). SCG, sodium cocoyl glutamate; SN, supernuclease; TKO, triple knockout; WT, wild-type.

Discussion

In this study, we produced GGTA1/CMAH/β4GalNT2 knock-out (TKO) pigs and successfully decellularized corneas harvested from these pigs using a novel method of 0.5% SCG combined with SN. Decellularized corneal stromal grafts were then implanted into rabbit corneas, where they demonstrated promising results. The key feature of this study was the use of corneas from TKO pigs. This study demonstrated the effectiveness of our novel decellularization method.

The three genes knocked out in our study are known to play a crucial role in the host’s graft rejection. The GGTA1 gene encodes an enzyme that synthesizes carbohydrate antigen galactose-alpha-1,3-galactose (α-Gal), a sugar molecule found on pig cells but not on human cells, which can trigger a strong immune response in humans, leading to hyperacute rejection of transplanted tissues (25,27,33). The CMAH gene is involved in the synthesis of N-glycolylneuraminic acid (Neu5Gc), a sialic acid variant present in most mammals but not in humans due to a mutation in the CMAH gene. When human immune cells encounter Neu5Gc, they recognize it as foreign, potentially leading to an immune response and rejection of the xenograft (13,25,27). The β4GalNT2 gene produces an enzyme that synthesizes the Sd antigen, another carbohydrate structure not present in humans. This Sd antigen can provoke an immune response when introduced into the human body (13,25,27).

The corneas from TKO pigs offer significant advantages in xenografts (25). These TKO corneas exhibit reduced immunogenicity, as the TKO significantly lowers the expression of antigens recognized by the human immune system, thereby decreasing the likelihood of hyperacute rejection (26,27). This reduction in immune response also minimizes inflammation, which is crucial for maintaining corneal transparency and preventing graft failure. Consequently, grafts have improved long-term survival rates, enhancing their compatibility with human recipients and potentially reducing the need for extensive immunosuppressive therapy. Overall, the use of TKO pig corneas in xenografts represents a significant advancement in reducing immunological barriers and improving success rates of corneal xenografts (25-27).

Decellularization is a process in which all cells and cellular components are completely removed through chemical, biological, or physical methods, leaving a biological scaffold of native extracellular matrix (ECM) proteins (24,30,34). The decellularization process is important for porcine cornea to be used as human grafts for several reasons. It reduces immunogenicity by removing cellular components and eliminating antigens that could trigger an immune response in the recipient, significantly lowering the risk of graft rejection (35). Additionally, by removing cellular debris and potential immunogenic substances, the process minimizes inflammation, which is essential for maintaining corneal transparency and preventing complications post-transplantation. However, an effective decellularization process should retain ECM components such as glycosaminoglycans and collagen to provide a scaffold that supports cell attachment and growth and to maintain corneal tensile strength, thus enhancing biocompatibility and promoting better integration with the recipient’s tissues. Protocols aimed at completely removing all cellular material inevitably can cause significant tissue disruption. Conversely, techniques that can preserve the ECM ultrastructure are likely to leave behind cellular artifacts and residual antigen molecules. Therefore, a balance between minimizing ECM ultrastructural disruption and sufficiently removing antigenic and immunogenic material is essential.

The rationale for combining SN with detergents like SLG or SCG was that while SN alone removes cell nuclei, it leaves behind lipid components, including cell membrane elements, which may cause a potential immunogenic response. Therefore, previous decellularization study has also recommended the use of detergents like SLG to destroy lipid components instead of using SN alone (28). As described in the results, decellularization using SN alone effectively reduced DNA content. However, we included SCG to further remove any remaining cell-derived lipid components. In this study, the SCG + SN decellularization method showed no visible nuclear component in the cornea tissue and did not show significant changes in collagen structures before and after decellularization through H&E staining. If significant damage had occurred to the ECM during the decellularization process, it might not be possible to maintain the transparent corneal graft observed in the rabbit experiments. However, the lack of detailed ultrastructural analysis of ECM scaffolds using electron microscopy might be a limitation of this study.

Research on new decellularization methods is important because most decellularization methods can cause alterations in tissue structure. Chemicals remaining in the decellularized tissue can promote inflammatory reactions when implanted into the body. The SCG-based decellularization method introduced in this study can effectively reduce DNA content while maintaining corneal transparency. Additionally, it can preserve corneal collagen and glycosaminoglycans. The tensile strength was found to be well-maintained after decellularization.

In our study, we observed excellent graft outcomes up to four weeks after transplanting TKO corneas or decellularized TKO corneas into rabbit corneas. Results were particularly notable compared to WT corneas. They were even superior to those obtained with decellularized WT stromal grafts. While decellularized WT corneas showed infiltration of inflammatory cells (CD3- or CD5-positive T cells) into the stromal graft, this was not observed in decellularized TKO corneas. It is well known that host immune response to donor tissue is the major reason of graft failure. Especially, T cells play a pivotal role in graft rejection. In previous studies, infiltration of host T cells and subsequent immune activation have been frequently observed in cases of rejected corneal xenotransplantation (36-39). These results suggest that decellularization using SCG and SN alone is not sufficient to effectively reduce the immunogenicity of donor tissues.

Additionally, as seen in comparisons of corneal neovascularization and opacity, TKO corneas demonstrated good graft outcomes up to four weeks, regardless of decellularization. Neovascularization and corneal opacity are well-known clinical manifestations of graft failure (11,40). Our results demonstrated that both the TKO method and the decellularization method could effectively inhibit graft rejection and inflammation. It was particularly interesting that the TKO method alone yielded good results, highlighting the importance of TKO in porcine xenografts, especially considering that decellularization did not show significant effects in WT corneas.

One of the major barriers in corneal xenotransplantation is the surgical procedure originated from cornea thickness and power mismatch between human recipients and porcine cornea. While normal human cornea thickness is around 540 µm, the thickness of pig cornea ranges from 666 to 995 µm depending on the age and breed of the pig used (33,41). In this study, we found that the corneal thickness of 2-month-old TKO pigs was 640 µm, which was suitable for suturing to a human recipient cornea. The central corneal power of the TKO pig was measured at 45.36 diopters, closely resembling that of a human cornea. Considering that the corneal power of wild-type porcine corneas has been previously reported to be around 40 diopters, the TKO cornea appears to be more suitable than wild-type porcine cornea for refractive power restoration (42).

Our study has several limitations. First, the survival rate of TKO pigs was not high. A and a significant number of newborns died. This issue has been commonly observed in genetically modified pigs. It is expected to be largely overcome with technological advancements. Second, the decellularization process of pig corneas we used needs optimization to consistently achieve effective results. The use of a single animal model, rabbits, is also a limitation of this study. Previous studies have reported results using non-human primates, which require a significant budget. Our research team also plans to conduct experiments using non-human primates in the near future. The 4-week postoperative observation period might be too short to draw firm conclusions. However, we plan to conduct longer-term observations using non-human primates in future experiments. In this study, the authors acknowledge that while the chosen SN and SCG combination decellularization method is new, it has the limitation of not being adequately compared with various previously introduced decellularization methods (28,30). Decellularization methods include chemical, physical, and biological approaches. The current study is significant as it proposes SCG as a new chemical option for decellularization. Our results indicate that SCG alone is insufficient for complete decellularization, necessitating the combination with SN. While SN alone effectively removes DNA content in the corneal stroma, it is a biological decellularization method that digests nucleic acid components (RNA and DNA only). Thus, SCG + SN is a combination method of chemical and biological decellularization.

Conclusions

In this study, we successfully decellularized corneas of TKO pigs using a novel SCG plus SN method and observed positive results after transplanting them into rabbit corneas. Corneal xenotransplantation is a promising solution to address the shortage of donor corneas. However, no research team has secured a porcine corneal graft that can be perfectly applied to humans. This underscores the need for ongoing research in this field and the importance of sharing research findings among researchers.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://atm.amegroups.com/article/view/10.21037/atm-25-3/rc

Data Sharing Statement: Available at https://atm.amegroups.com/article/view/10.21037/atm-25-3/dss

Peer Review File: Available at https://atm.amegroups.com/article/view/10.21037/atm-25-3/prf

Funding: This work was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the National Research Foundation of Korea (NRF), supported by the Ministry of Education (NRF-2021R1A2C1006087 to C.Y.P.), a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (RS-2023-KH135936 to C.Y.P.), and a grant from the Industrial Technology Innovation Program (20003915 to C.Y.P.), funded by the Ministry of Trade, Industry & Energy (MOTIE), Republic of Korea.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-25-3/coif). J.T.K. and P.H.C. are employees of MGENSolutions Co., Ltd. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All animal experiments were performed under a project license (IACUC reference number: 2019-1) granted by the Institutional Animal Care and Use Committee (IACUC) of the Biotechnology Research Institute of MGENSolutions Co., Ltd., Republic of Korea, in compliance with national guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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