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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 models^1,2. 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 setting^2,3. Among different tissues/organs, corneal explants, including that of humans^4,5,6, have been used broadly for ocular toxicity, irritation assessments^7,8, to study molecularly mechanisms underlying stem cell function⁹ and wound healing^10,11, 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 diseases^13,14. 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 healing^16,17. 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.

Protokół

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

1. Add penicillin-streptomycin to the Minimum essential medium (MEM) as supplements before use.
2. Prepare high-glucose culture media by adding 3.6 g of D-glucose to 1 L of supplemented MEM, which contains 5 mM glucose (equal to 90 mg/dL), to reach a 25 mM (equal to 450 mg/dL) final glucose concentration, mimicking hyperglycemia in diabetic patients.
3. Prepare 1% agarose in supplemented MEM with 5 mM or 25 mM glucose by adding 0.2 g agarose to 20 mL of MEM and heating it in a microwave until the agarose dissolves.
4. Transfer the agarose-containing solution into a water bath maintained at 48 °C.
5. Before the experiment, autoclave all experimental reagents, such as distilled water, PBS, and surgical equipment: hemostat, forceps, scalpel handle, scissors, and trephine, as well as beakers, paper towels, lint-free wipes, gloves, and pipette tips. Soak the silicon mold, razor blade, and blade holder in 70% alcohol for 30 min and wash with sterile PBS three times.

2. Porcine eyeball preparation for corneal culture

1. Obtain porcine eyeballs from a local abattoir and transport them to the laboratory on ice in a moist chamber.
   NOTE: Bovine eyes can also be used in a similar fashion. However, bovine eyes and corneas differ from human eyes/corneas more remarkably. For example, human and porcine corneas have 5-7 layers of epithelial cells, while bovine corneas have about 20 layers^13,14. Moreover, bovine eyes are more likely to be contaminated during corneal organ culture; hence, more bovine eyes are needed for statistical analysis.
2. Place the porcine eyes in a 1 L beaker containing sterilized PBS.
   NOTE: The porcine eyeballs are collected within 1 h after slaughter and transported to the lab in about 2 h. Overall, the eyes were used within 4 h (step 2.3).
3. Hold an eyeball with tweezers and remove the extraocular tissues with a scissor in a sterile Petri plate.
4. Rinse the eyeballs with distilled water once and PBS twice.
5. Rinse the eye bulbs with a Povidone-Iodine antiseptic solution for 10 s, followed by sterilized PBS three times washing.
6. Incubate the eyeballs in PBS containing 20 µg/mL gentamicin for 30 min and rinse them twice with PBS.

3. Epithelium wounding

1. Hold an eyeball with sterilized lint-free wipes and mark the center of the corneas with a 6 mm trephine.
2. Gently scrape epithelial cells within the trephine-marked circle with a small scalpel or corner-blunted soft razor blade, remove all cell debris but leave the basement membrane intact, and clean the wound area with cotton swabs.
   NOTE: The scalpel with collected epithelial cells can be transferred to a microcentrifuge tube placed on ice. The cells can be lysed immediately or stored in a -20 °C freezer to serve as the controls..

4. Corneal organ culture and ex vivo hyperglycemia modeling

1. Dissect the eyeball by cutting along corneal-scleral rims with a scalpel and scissors and rinse the corneas in a sterilized 1000 mL beaker containing PBS (pH 7.4).
2. Place the excised corneas upside down into a sterile mold made from non-toxic liquid mold-making silicone.
   NOTE: Pour the non-toxic silicone mold-making liquid into the holes of a 50 mL tissue culture/test tube rack, using the bottom of the 30 mL ultracentrifuge tube to make a half-round mold.
3. Fill the endothelial corneal cavity with MEM containing 1% agarose maintained at 48 °C and allow the mixture to gel at the room temperature..
4. Invert and transfer the corneas to a 35 mm dish, and add 2 mL of MEM with or without testing agent(s) dropwise to the surface of the central cornea to cover the limbal conjunctiva region, leaving the epithelium exposed to the air (Figure 1).
5. Place the culture dishes in a humidified 5% CO₂ incubator at 37 °C and replace the media with fresh MEM media with or without a testing agent(s) daily for 2 days.
6. Establish an ex vivo hyperglycemia model by adding L-glucose to MEM to reach a total of 25 mM glucose, which is used for 1% agarose gel preparation and as culture media.

5. Corneal function assessment

1. Determine the epithelial wound healing rate by staining the wounded corneas with Richardson's staining20 to mark the remaining wound area and photograph it (Figure 2 and Figure 3). Quantify the wound size with Image-J or Photoshop software (histogram).
2. Process the corneas for histology, histochemistry, and immunohistochemistry analyses (Figure 2 and Figure 3) by embedding them in the OCT and cryostat sectioning. Fix the sections with ice-cold acetone, stain with H&E staining, or block with 1% BSA for 1 h for TUNEL staining or immunohistochemistry to detect proteins and signaling molecules (such as phosphorylated Erk and ATK).
   NOTE: Most commercially available antibodies were not tested for porcine antigens. However, if an antibody recognizes both human and mouse antigens, it can mostly be used to detect the corresponding porcine antigen Western blotting and immunohistochemistry.
3. Wash the stained corneas with PBS and use the same-sized trephine to mark the original wound and scrap epithelial cells within the marker circles.
   1. Collect epithelial cells with a small scalpel, immerse the scalpel with collected cells a centrifuge tube precooled on ice.
   2. Store the collected cells in -80 °C deep freezer or extract them immediately and perform lysis for Western blotting and/or ELISA or in RNA extraction buffer for PCR (if required)^10,16,21.

Wyniki

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 a...

Dyskusje

Cultured bovine and mostly porcine corneas have been used to assess the toxicities of cosmetic chemicals, glaucoma medications, and nonsteroidal anti-inflammatory drugs^21,26. 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 h...

Materiały

Name	Company	Catalog Number	Comments
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