porcine corneal organ culture and epithelial wound healing for chemical toxicity tests

Ex Vivo Porcine Corneal Culture System for Toxicity Testing and Wound Healing

While cell models possess limited clonal populations and fail to reproduce an organism&#39;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&#39;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,&#160;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-1&#946; versus IL-1Ra18 and TGF&#946;3 versus TGF&#946;119 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.

Author Spotlight: Exploring Corneal Innate Immunity and Delayed Wound Healing in Diabetic Patients

Evaluating Therapeutic and Chemical Toxicity Using Organ-Cultured Porcine Corneas and Epithelial Wound Healing

We are investigating cornea innate immunity and diabetic wound healing. Specifically, we aim to understand why diabetic patients are more susceptible to bacterial infection and what causes delayed wound healing associated with hypoglycemia. We've identified that the balance between IL-1 beta and IL-1 receptor antagonist, as well as TGF Beta-1 and TGF Beta-3, is important for corneal wound healing.This could lead to a new treatment for diabetic care toxic. Our model may make human-delayed wound healing in diabetic patients. This allows us to study, program the cell death pathway, and develop targeted therapies.

Porcine Corneal Organ Culture and Epithelial Wound Healing for Chemical Toxicity Tests

To begin, take porcine eyes placed in a one-liter beaker. Hold an eyeball with tweezers and remove the extra ocular tissues with scissors in a sterile Petri plate. For epithelium wounding, hold the eyeball using sterilized lint-free wipes and mark the center of the cornea with a six-millimeter trephine.Then using a small scalpel or corner-blunted soft razor blade, gently scrape the epithelial cells within the trephine-marked circle. Remove all cell debris while ensuring the basement membrane remains intact. Afterward, clean the wound area with cotton swabs.Using a scalpel and scissors, dissect the eyeball by cutting along the corneo-scleral rims. Then rinse the corneas in a sterilized 500-milliliter beaker containing PBS. Next, place the excised corneas upside down into a sterile mold.Fill the endothelial corneal cavity with a minimum essential medium, or MEM, containing 1%agarose maintained at 48 degrees Celsius. Now, invert and transfer the corneas to a 35-millimeter dish. Add two milliliters of MEM with or without the testing agent dropwise to the surface of the central cornea, ensuring the limbal conjunctiva region is covered while leaving the epithelium exposed to air.Then place the culture dishes in a humidified 5%carbon dioxide incubator at 37 degrees Celsius. At 40 hours post wound, stain the wounded cultured corneas with Richardson staining solution. Wash the stained corneas with PBS before photographing them.Then use the same size trephine to mark the original wound, and using a small scalpel, scrape the epithelial cells within the marked circles. Remove the collected cells and transfer them into a pre-cooled centrifuge tube. The remaining wound area was significantly larger in corneas treated with nepafenac 0.1%compared to those treated with bromfenac 0.09%and ketorolac 0.45%Corneas treated with LL-37 at 0.2 and 0.5 micrograms per milliliter showed significantly accelerated wound healing under high-glucose conditions compared to untreated corneas.

porcine-corneal-organ-culture-epithelial-wound-healing-for-chemical

Research

JoVE Journal

Medicine

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 &#176;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.

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.