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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board
1. Preparation of an inoculum
<ol>
	<li>Pour 10 mL of LB broth into a 50 mL conical flask with a foam stopper.</li>
	<li>Transfer a colony of&#160;P. aeruginosa&#160;strain PAO1 or strain PA14 from a fresh agar plate and incubate at 37 &#176;C for 3-4 h until the bacteria are in mid-log phase.</li>
	<li>Transfer the culture of bacteria to a 50 mL tube and centrifuge at 3,000 x&#160;g&#160;for 5 min. Remove the supernatant and re-suspend the cell pellet in PBS.</li>
	<li>Repeat step 1.3 two more times to wash the cells. Resuspend the cell pellet in PBS and adjust the optical density at 600 nm to approximately 0.6 using sterile PBS as a blank.</li>
</ol>
2. Infecting the corneoscleral button
<ol>
	<li>Remove media from the Petri dish and rinse corneas twice with 1 mL of sterile PBS.</li>
	<li>Gently squeeze forceps while holding the cornea in-between. Use a 10A scalpel to make four cuts&#8211;two vertical, two horizontal-in the central section of the corneoscleral button through the epithelial layer to the underlying stroma.</li>
	<li>Place a sterile glass mold in a 6-well plate with the wide part up and place the cornea in the middle of the glass mold, epithelium side facing down. Make the cut right in the center of the bottom part of the glass mold.</li>
	<li>CRITICAL STEP: Pour 1 mL of 1% (w/v) low melting point agar dissolved in DMEM to fill the glass mold with cornea completely.</li>
	<li>Allow the agar to set and then invert the glass mold so that the corneal epithelium is facing upwards.</li>
	<li>Pipette 15 &#956;L of the bacterial culture with OD600nm&#160;= 0.6 (for&#160;P. aeruginosa&#160;this equates to approximately 1 x 107&#160;colony forming units (CFU) in 15 &#956;L) directly into a cut area and then add 85 &#956;L of PBS to the top to keep the corneal epithelium moist.&#160;Dilute the remaining bacterial culture and plate on agar to count colony forming units for the inoculum.</li>
	<li>Add 1 mL of DMEM without antibiotics to the bottom of each well with the glass mold. Incubate the 6-well plate with the infected corneoscleral buttons in a humidified incubator at 37 &#176;C with 5% CO2&#160;for up to 24 h.</li>
	<li>Set up uninfected control cornea alongside every experiment. To set up uninfected control, replace the 15 &#956;L of bacterial culture in step 2.6 with sterile PBS.</li>
</ol>

<table><tbody><tr><td>50 mL Falcon tube</td><td>SLS</td><td>352070</td><td></td></tr><tr><td>Amphotericin B</td><td>Sigma</td><td>A2942</td><td></td></tr><tr><td>Cellstar 12 well plate</td><td>Greiner Bio-One</td><td>665180</td><td></td></tr><tr><td>Dextran</td><td>Sigma</td><td>31425-100mg-F</td><td></td></tr><tr><td>Distel</td><td>Fisher Scientific</td><td>12899357</td><td></td></tr><tr><td>DMEM + glutamax</td><td>SLS</td><td>D0819</td><td></td></tr><tr><td>Dual Oven Incubator</td><td>SLS</td><td>OVe1020</td><td>Sterilising oven</td></tr><tr><td>Epidermal growth factor</td><td>SLS</td><td>E5036-200UG</td><td></td></tr><tr><td>F12 HAM</td><td>Sigma</td><td>N4888</td><td></td></tr><tr><td>Foetal calf serum</td><td>Labtech International</td><td>CA-115/500</td><td></td></tr><tr><td>Forceps</td><td>Fisher Scientific</td><td>15307805</td><td></td></tr><tr><td>Heracell VIOS 160i</td><td>Thermo Scientific</td><td>15373212</td><td>Tissue culture incubator</td></tr><tr><td>Insulin, recombinant Human</td><td>SLS</td><td>91077C-1G</td><td></td></tr><tr><td>LB agar</td><td>Sigma</td><td>L2897</td><td></td></tr><tr><td>Multitron</td><td>Infors</td><td>Not appplicable</td><td>Bacterial incubator</td></tr><tr><td>PBS</td><td>SLS</td><td>P4417</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>SLS</td><td>P0781</td><td></td></tr><tr><td>Petri dish</td><td>Fisher Scientific</td><td>12664785</td><td></td></tr><tr><td>Petri dish 35x10mm CytoOne</td><td>Starlab</td><td>CC7672-3340</td><td></td></tr><tr><td>Povidone iodine</td><td>Weldricks pharmacy</td><td>2122828</td><td></td></tr><tr><td>Safe 2020</td><td>Fisher Scientific</td><td>1284804</td><td>Class II microbiology safety cabinet</td></tr><tr><td>Scalpel blade number 15</td><td>Fisher Scientific</td><td>O305</td><td></td></tr><tr><td>Scalpel Swann Morton</td><td>Fisher Scientific</td><td>11849002</td><td></td></tr></tbody></table>

porcine ex vivo cornea model of bacterial keratitis: a technique to establish bacterial infection in corneal epithelial cells

Source: Okurowska, K., et al. <a href="https://www.jove.com/v/61156">Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis.</a> J. Vis. Exp (2020).
This video describes a protocol to set up an ex vivo model for bacterial keratitis using a corneoscleral button dissected from the porcine eye. Inoculated bacteria traverse the corneal epithelial incisions to invade the underlying stroma and establish an infection within the corneal tissues that mimic the in vivo infection.

Porcine Ex vivo Cornea Model of Bacterial Keratitis: A Technique to Establish Bacterial Infection in Corneal Epithelial Cells

Source: Okurowska, K., et al. Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis. J. Vis. Exp (2020).
...

In the vertebrate eye, the cornea - the outermost protective covering- is infected by Pseudomonas aeruginosa, resulting in a corneal inflammatory condition - keratitis.&#160;
To establish an ex vivo corneal keratitis model, place a porcine eye corneoscleral button in a Petri dish. This tissue segment consists of the cornea surrounded by remnants of sclera - the fibrous eye covering. Supplement the Petri dish with antibiotic-containing media to eliminate contaminating microbes from the corneal epithelial surface.
Locate the central portion of the cornea. Make superficial horizontal and vertical incisions in the desired pattern piercing the outermost corneal epithelial layer. Transfer the incised tissue, cornea side down, into a specialized hollow mold.&#160;
Fill the corneal cavity with liquified agar and allow to solidify, restraining the tissue inside the mold. Invert the mold to expose the wounded surface of the corneal epithelium. Pipette the Pseudomonas suspension into the incised areas of the cornea. Overlay the mold with more growth medium for optimal bacterial growth. Supplement the culture dish with media and incubate.&#160;
Bacteria traverse the epithelial incision to invade the stroma. Inside the stroma, bacteria cause the infiltration of immune cells, proteins, and fluid in the cornea resulting in an inflammatory response. The fluid build-up increases the corneal opacity, confirming bacterial keratitis.

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Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Biology

Large Animal Models

Porcine

1.0K Views. Source: Okurowska, K., et al. Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis. J. Vis. Exp (2020). This video describes a protocol to set up an ex vivo model for bacterial keratitis using a corneoscleral button dissected from the porcine eye. Inoculated bacteria traverse the corneal epithelial incisions to invade the underlying stroma and establish an infection within the corneal tissues that mimic the in vivo infection. 

Video: Porcine Ex vivo Cornea Model of Bacterial Keratitis: A Technique to Establish Bacterial Infection in Corneal Epithelial Cells - Concept

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential to establish an ex vivo model that can evaluate SIM performance accurately, for developing high-performance SIMs. In our previous study, we developed a new ex vivo model that can be used to evaluate the performance of various SIMs in detail by applying constant tension to the specimen&#39;s ends. We also confirmed that the proposed new ex vivo model allows accurate submucosal elevation height (SEH) measurement under uniform conditions and detailed comparisons of the performances of various types of SIMs. Here, we describe the new ex vivo model and explain the detailed setup methodology of this model. Since all parts of the new model were easy to obtain, the setup of the new model could be completed quickly. SEH of various SIMs could be measured more accurately by using the new model. The critical factor that determines SIM performance can be identified using the new model. SIM development speed will drastically increase after the factor has been identified.

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

We developed a new ex vivo model that applies constant tension to the porcine gastric specimen. This development made it possible to evaluate the performance (the height and duration of the submucosal elevation) of various SIMs accurately.&#160; The detailed setup methodology of this new model is explained.

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential ...

JoVE Journal

Bioengineering

Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals

Viruses and bacteria can cause a variety of ocular surface defects and degeneration such as wounds and ulcers through corneal infection. With a seroprevalence that ranges from 60-90% worldwide, the Herpes Simplex Virus type-1 (HSV-1) commonly causes mucocutaneous lesions of the orofacial region which also manifest as lesions and infection-associated blindness. While current antiviral drugs are effective, emergence of resistance and persistence of toxic side-effects necessitates development of novel antivirals against this ubiquitous pathogen. Although in vitro assessment provides some functional data regarding an emerging antiviral, they do not demonstrate the complexity of ocular tissue in vivo. However, in vivo studies are expensive and require trained personnel, especially when working with viral agents. Hence ex vivo models are efficient yet inexpensive steps for antiviral testing. Here we discuss a protocol to study infection by HSV-1 using porcine corneas ex vivo and a method to treat them topically using existing and novel antiviral drugs. We also demonstrate the method to perform a plaque assay using HSV-1. The methods detailed may be used to conduct similar experiments to study infections that resemble the HSV-1 pathogen.

We describe the use of a porcine cornea to test the antiviral efficacy of experimental drugs.

Viruses and bacteria can cause a variety of ocular surface defects and degeneration such as wounds and ulcers through corneal infection. With a ...

Immunology and Infection

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for ...

Porcine Ex vivo Cornea Model of Bacterial Keratitis: A Technique to Establish Bacterial Infection in Corneal Epithelial Cells

Transcript

Porcine Ex Vivo Cornea Model of Bacterial Keratitis: A Technique to Establish Bacterial Infection in Corneal Epithelial Cells

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons