Porcine corneal ex vivo organ culture and epithelial wound healing provide an economical, ethical, reproducible, and quantitative means for testing the ocular toxicity of chemicals. They also aid in elucidating mechanisms underlying the regulation of epithelialization and tissue repair, and in evaluating therapeutics for treating diabetic keratopathy and delayed wound healing.
Due to its anatomical and physiological similarities to the human eye, the porcine eye serves as a robust model for biomedical research and ocular toxicity assessment. An air/liquid corneal culture system using porcine eyes was developed, and ex vivo epithelial wound healing was utilized as a critical parameter for these studies. Fresh pig corneas were processed for organ culture, with or without epithelial wounding. The corneas were cultured in a humidified 5% CO2 incubator at 37 °C in MEM, with or without testing agents. Corneal permeability and wound healing rates were measured, and epithelial cells and/or whole corneas can be processed for immunohistochemistry, western blotting, and qPCR for molecular and cellular analyses. This study describes a detailed protocol and presents two studies using this ex vivo system. The data show that porcine corneal organ culture, combined with epithelial wound healing, is a suitable ex vivo model for chemical toxicity testing, studying diabetic keratopathy, and identifying potential therapies.
While cell models possess limited clonal populations and fail to reproduce an organism's in vivo architecture, organ culture or explant offers insights into organ function, development, disease mechanisms, and potential therapies while providing ethical and physiological advantages over other experimental models1,2. In addition to reducing the number of animals needed, culturing explants control and sensibly manipulate the surrounding conditions, which is ideal for a detailed exploration of factors controlling cell proliferation, migration, wound response, and cellular differentiation in an organ culture setting2,3. Among different tissues/organs, corneal explants, including that of humans4,5,6, have been used broadly for ocular toxicity, irritation assessments7,8, to study molecularly mechanisms underlying stem cell function9 and wound healing10,11, and to Primary open-angle glaucoma12.
Porcine corneas share several structural and physiological similarities with human corneas, making them an excellent model for studying human corneal biology and diseases. Structurally, both have Bowman's layer, 5-7 layers of epithelial cells, and similar curvature and diameter. Physiologically, they are highly transparent, have similar tear film composition and corneal hydration, exhibit comparable patterns and functions of corneal innervation, and follow similar wound healing processes, making them an excellent model for studying human corneal biology and diseases13,14. While human and porcine corneas have slight differences in collagen fibril arrangement and water content, their immune signaling and responses are not identical. These differences pose challenges for xenotransplantation15. Hence, species-specific differences must be considered while interpreting experimental data.
Compared to human corneas, porcine eyes are readily available as byproducts of the meat industry, making them cost-effective and easily accessible for research14. Using porcine corneas helps reduce the need for human donor corneas and minimizes the ethical concerns associated with animal testing. Moreover, the availability of many porcine corneas at one time allows for consistent and reproducible experiments, which is crucial for reliable research outcomes.
A porcine corneal organ culture system was initially used to replace animal tests of cosmetic chemicals and ocular drugs7. This system has been used to study corneal epithelial wound healing and to identify several important signal pathways such as HB-EGF ectodomain shedding, lipid mediator lysophosphatidic acid stimulation, and EGFR activation for corneal wound healing16,17. Using high glucose as a pathologic factor, an ex vivo model of hyperglycemia was established with delayed epithelial wound healing to mimic human diabetic keratopathy. Using this model, the balance expressions of IL-1β versus IL-1Ra18 and TGFβ3 versus TGFβ119 were shown to be important factors for proper wound healing in the corneas, and manipulation of these balances may be used to treat diabetic keratopathy. Hence, porcine organ culture represents a relevant, economical, and manipulating experiment system with various applications in chemical toxicity tests, biomedical research, drug discovery, and assessing tissue damage and repair in response to ocular exposure to chemical weapons.
In this article, a detailed protocol of porcine corneal organ culture is described, and its applications for assessing the potential effects of ocular NSAID (NS) eye drops on corneal health and for determining signaling pathways and biological processes involved in the pathogenesis of diabetic keratopathy are illustrated.
Since fresh pig corneas are a byproduct of the food industry, the Institutional Animal Care and Use Committee did not need to approve their use for research. Unlike human corneas used in research, there are no biohazard concerns, and unused parts of pig eyes can be disposed of as regular garbage. The reagents and equipment used for this study are listed in the Table of Materials.
1. Preparation for organ culture
2. Porcine eyeball preparation for corneal culture
3. Epithelium wounding
4. Corneal organ culture and ex vivo hyperglycemia modeling
5. Corneal function assessment
Cataract surgery is one of the most frequently performed procedures globally, and eye drops play a crucial role in post-surgery care. Applying eye drops after cataract surgery helps prevent complications such as eye infections, inflammation, and macular edema. NSAID (NS) eye drops, including ketorolac, bromfenac, and nepafenac, have commonly been used to treat pain and swelling of the eye before, during, and after cataract surgery. The long-term use of these eye drops that contain various amounts of preservatives, such as benzalkonium chloride, may have adverse effects on the health of the corneas22.
Using pig corneal organ culture, the effects of these NS eye drops on the rate of epithelial wound healing were assessed (Figure 2). Pig corneas were processed for organ culture as described in the protocol section, and culture in MEM media with or without NS eye drops applied as described in Figure 1. Corneas cultured in MEM medium alone as the control (without BAK) had the mean (SD) of remaining wound areas at 48 hours presented as 565 ± 1263 pixels while treated corneas had 47,322 ± 13,736 pixels for Nepafenac 0.1% (0.05% BAK), 29,093 ± 14,295 pixels for bromfenac 0.09% (0.005% BAK), and 29,093 ± 14,295 pixels for ketorolac 0.45% (0% BAK), respectively.
The remaining wound areas were notably smaller in corneas treated with ketorolac 0.45% than those treated with nepafenac 0.1% (P < 0.01) or bromfenac 0.09%. Additionally, corneas treated with nepafenac 0.1% had a remarkably larger mean remaining wound area compared to those treated with bromfenac 0.09% (P < 0.01). There were no significant differences between the mean of the control and ketorolac 0.45% treated corneas. The mean of the remaining wound area of the corneas appeared to be related to the concentration of BAK. Hence, reducing BAK concentrations or total abandonment as a preservative has been the trend for developing new or improving existing eye drops and medications.
Using cultured porcine corneas as an ex vivo model to study epithelial wound healing, it was found that epithelial wound closure was highly impaired in corneas cultured under high glucose conditions (25 mM glucose) compared to those cultured in normal glucose (5 mM glucose) or high mannitol (5 mM of glucose containing 20 mM of D-mannitol, used as an osmotic control)10. For instance, the ability of LL-37, a peptide secreted by epithelial and immune cells from the gene cathelicidin, to rescue wound healing delayed by high glucose was tested in porcine corneas cultured under normal or high glucose conditions. Corneas cultured in normal glucose completely healed a 4 mm wound within 48 h, while those in high glucose conditions exhibited significantly slower wound closure. LL-37 at concentrations of 0.2 µg/mL and 0.5 µg/mL significantly accelerated wound healing that was delayed by high glucose. Using this model, the involvement of EGFR signaling10, IL-1Ra18, and TGFβ isoforms19 in promoting corneal epithelial wound healing was reported in pig corneas cultured in 25 mM glucose. These results were confirmed in mouse models of type 1 and/or type 2 diabetes19,23,24. Hence, studies combining ex vivo models of hyperglycemia and in vivo, mouse models of diabetes not only greatly reduce the number of live animals needed but also allow the large-scale screening of agents, alone or in combination, for their ability to improve diabetic wound healing, which is usually impaired in diabetic patients with an enormous amount of emotional, social, and economic burdens to the patient's family and the society.
Figure 1: Diagram of the corneal organ culture model for chemical toxicity tests. The convex shape of the cornea was maintained by an agarose/collagen gel in MEM, which supports the overlying endothelial cells. To cover the limbal region, drop by drop the culture medium was added to the center of the cornea. The dissolved test chemicals in the culture medium were also applied drop by drop to ensure thorough wetting of all surfaces. Please click here to view a larger version of this figure.
Figure 2: Corneal epithelial wound healing in cultured porcine corneas treated with NS eye drops. (A) Representative images of epithelial wound closure induced by NS eye drops in cultured porcine corneas. A 4 mm diameter epithelial wound was created and allowed to heal for 48 h in MEM (Control); 2-3 eye drops were applied to the cultured corneas every other hour during the daytime. Richardson staining solution was used to stain the wounded corneas to reveal the initial wound and the remaining wound areas in both the control (MEM) and NS-treated corneas. (B) Evaluation of wound healing in cultured porcine corneas treated with different NS. The extent of wound coverage (0% for no migration and 100% for complete coverage of the wound bed) was determined as described in the protocol section. Data are presented as mean ± SD of at least five corneas. Please click here to view a larger version of this figure.
Figure 3: LL-37 reduces high glucose-mediated delay in epithelial wound healing in porcine corneal organ culture. The epithelial wound of 5 mm diameter was made (A, original wound) in the center of porcine corneas and allowed to heal for 48 h in MEM containing NG (B; 5 mM d-glucose), or high glucose (C; 25 mM d-glucose) in the presence of 0.2 µg/mL (D) or 0.5 µg/mL (E) LL-37. Organ-cultured corneas were stained with Richardson staining solution to show the remaining wounds. Micrographs represent 1 of 3 samples performed each time. The bar graph represents the statistical analysis of the extent of healing. Values are expressed as mean ± SEM. *P < 0.05 and **P < 0.01 (Student's t-test) compared with HG. Results are representative of five independent experiments. This figure is adapted from Yin et al.25. Please click here to view a larger version of this figure.
Cultured bovine and mostly porcine corneas have been used to assess the toxicities of cosmetic chemicals, glaucoma medications, and nonsteroidal anti-inflammatory drugs21,26. Pig corneas have also been used as an ex vivo model of human diabetic keratopathy. Unlike rabbit eyes, the pig eye resembles the human eye anatomically, physiologically, and biomechanically27, hence, being used for xenotransplantation into human15, including the cornea, liver, heart, and kidney. As such, the data generated using pig corneas should be more relevant to human exposure. Moreover, pig eyes are fresh, inexpensive, resistant to microbial contamination or infection during organ culture, and provide a relatively large amount of biological materials for molecular and cellular biology analyses. Hence, the pig corneal organ culture is an excellent ex vivo model with a broad application in biomedical research and in chemical toxicity determination.
Preventing the cultured corneas from microbial contamination is critical to this protocol’s success. In addition to thoroughly washing, treatment with a Povidone-iodine antiseptic solution and incubation with gentamicin for 30 min are important steps to minimize microbial contamination.
Although the organ culture maintains the structural integrity of tissues, the immune and neuronal elements are lacking. This is the major limitation of ex vivo studies. In many cases, organ culture or explant can be used as a complement to animal study. However, cosmetics chemicals must evaluate the ocular irritancy for their safe handling and use before releasing into the market28,29. The Draize rabbit eye irritation test, developed in 1944, has long been considered a gold standard for assessing eye irritation. However, it has faced criticism for animal welfare concerns due to its invasive and distressing procedures30. Animal testing has been banned. There is a great need for alternatives to animal testing for safety and efficacy testing of cosmetic products and cosmetic ingredients. Combining corneal organ culture with measurements of corneal epithelial permeability after chemical exposure holds promise as a mechanistically based alternative to in vivo animal testing30. Hence, using this ex vivo system may fulfill the goal of the 3Rs, particularly for testing cosmetics and newly produced chemicals for ocular toxicity and irritation8.
This ex vivo pig organ culture system was also used to compare the potential adverse effects of topical NS and demonstrated that removal of BAK in ketorolac 0.45% ketorolac 0.45% had statistically less impact on corneal re-epithelialization than prior ketorolac formulations (0.4% and 0.5%)31, bromfenac 0.09%, and nepafenac 0.01% (Figure 2). Several formulations developed by a company to increase the permeability of an IOP-lowering drug were also assessed for corneal toxicity, allowing ruling out certain formulations without further testing in animals and/or patients.
The studies using ex vivo models featuring high versus normal glucose cultures provide data complementary to mouse models of diabetic keratopathy and delayed wound healing (Figure 3). Using this combined approach, HB-EGF ectodomain shedding and EGFR activation10,16, exogenous lysophosphatidic acid17, and TGFβ3, but not β119,32 hasten delayed epithelial wound healing in diabetic corneas were demonstrated and, therefore, might be used to treat diabetic keratopathy. In addition, ex vivo organ culture can also be used to determine the dosage and time course of a tested reagent, providing experimental bases for in vivo study with fewer animals to be used. Hence, high glucose in organ-cultured pig corneas may be used for studying underly mechanisms for diabetic complications and testing reagents for their ability to promote delayed corneal wound healing by hyperglycemia16,17. The results shown in Figure 3 indicate that the antimicrobial peptide LL-37 partially mitigated the impaired wound healing caused by high glucose (HG) in an EGFR- and PI3K-dependent manner, and restored EGFR signaling disrupted by HG in cultured porcine corneas. High glucose conditions reduced LL-37 expression in cultured human corneal epithelial cells (HCECs). Thus, LL-37 acts as a tonic factor that promotes EGFR signaling and enhances epithelial wound healing under both normal and high glucose conditions. Given its antimicrobial and regenerative properties, LL-37 may be a promising therapeutic option for diabetic keratopathy. Other factors such as microRNAs, antioxidants, ER stress inhibitors, and necroptosis inhibitors might also be tested in this system for their ability to overcome the negative effects of hyperglycemia on corneal health and for providing information for establishing a network of pathways and biological processes involved in corneal wound healing and their defects in diabetic corneas.
There is renewed interest in developing medical countermeasures to reduce mortality and serious morbidity during and after major public health emergencies involving the deliberate or accidental large-scale release of highly toxic chemicals (HTCs), such as vesicants28,29,33. This ex vivo pig corneal culture model with filter papers wetted with nitrogen mustard as a means of ocular exposure nitrogen mustard was demonstrated to cause corneal destruction in a dosage and exposure-time-dependent manner. DNA alkylation and cross-linking were the major causes of cell damage (Yu et al., unpublished). Pig organ culture is an ideal and complementary model to animal models for this line of studies of chemicals used as weapons, as they cause several ocular damages, even blindness, in humans and test animals.
The authors have nothing to disclose.
We thank Drs. Keping Xu (M.D. and O.D.) and Jia Yin (M.D. and Ph.D.) for their contributions to the development of bovine and porcine corneal organ culture and Ray Guo and Andy Wu of Troy High School for the artwork of Figure 1. Dr. Yu's lab research was funded by NIH grants (R01 EY010869, R01EY035785, P30 EY04068) and by Research to Prevent Blindness at Kresge Eye Institute.
1.7 mL tubes | Axygen | AXYMCT175SP | |
Agarose | Thermo Scientific | R0491 | |
Bromfenac (Prolensa) 0.09% | |||
Camera | Canon | PowerShot A620 | |
Cell Culture Dish | Corning | 430165 | |
D-glucose | Sigma | 50-99-7 | |
Dissecting microscope | Zeiss | Stemi 2000c | |
Forceps | FisherScientific | 10-316A | |
Hemostat | FisherScientific | 13-812-14 | |
Ketorolac (Acular) 0.45% | Kresge Clinic | ||
Kimwipes | Kimtech | 34155 | |
LL-37 | Tocris | 5213/1 | |
Minimum essential medium (MEM) | Gibco | A1048901 | |
Nepafenac (Ilevro) 0.1% | |||
Penicillin-streptomycin | Gibco | 15070063 | |
Phosphate buffered saline | Sigma | P4417 | |
Pig eyes | Bernthal Packing Inc. | ||
Pipet tips | VWR | 76322-164 | |
Porcine corneas | Bernthal Packing , Inc. Frankenmuth, MI | ||
Povidone-Iodine | Betadine | ||
Q-Tips cotton swabs | Q-Tips | ||
Razor blade | Electron Microscopy Sciences | 72002-01 | |
Razor blade holder | Stotz | ||
Scalpel | Bard-Parker | 377112 | |
Scalpel Handle | Bard-Parker | #3 | |
Scissors | FisherScientific | 08-951-20 | |
Silicon mold | |||
Tissue culter enclosure | Labconco | 5100000 | |
Trephine | Acu.Punch | 3813775 | |
Water bath | VWR | 1235 |