This work was supported by NIH-NEI R01 EY024942, Research to Prevent Blindness, Upstate Medical University Unrestricted Research Funds, and Lions District 20-Y. Microscopy and image analysis of paraffin sections were performed at the Microscopy CORE and histological slide preparation was performed at the Biorepository and Pathology CORE at the Icahn School of Medicine at Mount Sinai.

1. Organ Culture
<ol>
	<li>Preparations
	<ol>
		<li>Prepare agar solution as follows. In a small flask, prepare 1% agar and 1 mg/mL bovine collagen in DMEM-F12 up to 20 mL. Bring to boil on a hot plate. Put the solution into a 50 mL conical tube. Place tube in a water bath on a hot plate to keep the solution from solidifying.</li>
		<li>Prepare supplemented serum-free media (SSFM) as per the composition provided in the Table of Materials. 
		NOTE: The necessary amount of SSFM to be prepared depends on the number of corneas to be processed. Usually 30 mL is enough media for 4 corneas.</li>
	</ol>
	</li>
	<li>Dissection 
	NOTE: Perform this step in a dissection hood or a chemical hood. The eyes are shipped with lids still attached in individual bags to protect the globes.
	<ol>
		<li>Remove globes from lids with a straight-edge surgical blade on an ethanol-cleaned chopping board. Remove excess fatty tissue from the eye using either a blade or a small scissors (Figure 3A, 3B).</li>
		<li>After removing the globe from the lid, hold the globe posteriorly with forceps and immediately dip the eye in phosphate-buffered saline (PBS). Quickly dip it 3x in 10% iodine (in a 100 mL beaker). Quickly dip 2x in PBS (~100 mL in a beaker, change PBS frequently).</li>
		<li>Using a clean towel or tissue, wrap the eye circumferentially with enough pressure to have a taut corneal surface to cut with the trephine. 
		NOTE: Take care to prevent the towel or tissue from contacting the cornea.</li>
	</ol>
	</li>
	<li>Wounding
	<ol>
		<li>Use a 6 mm trephine to wound the center of the cornea. Penetrate the epithelium and anterior stroma without making a full-thickness wound through the entire cornea. 
		NOTE: If the endothelium is penetrated, a loss of pressure and leaking fluid will be seen. In this case the eye should be discarded.</li>
		<li>Place the trephine in the center of the cornea, rotate it 180&#176; clockwise and counter-clockwise 5x (each time the direction is changed it will count as one time) while applying light pressure to deepen the wound. 
		NOTE: The wound should now be deep enough to allow a tissue flap to be lifted using a pair of forceps. If this is not the case repeat step 1.3.2.</li>
		<li>Lift the flap from the edges. At the same time, either with the other hand or with a second person, use a blade, cutting parallel to the globe to cut away the tissue as the forceps continue to lift off the anterior cornea within the wound margin. At the conclusion of this step there should be a circular wound located at the center of the cornea (See Figure 3C, 3D).</li>
	</ol>
	</li>
	<li>Cutting and Removing the Cornea from the Globe
	<ol>
		<li>Holding the eye with the tissue, make a small incision 1 mm away from the edge of the cornea with a blade so that the limbus is included in the organ culture.</li>
		<li>Using small, sharp scissors access the incision created in the prior step to cut around the globe, keeping a millimeter margin throughout the cornea to keep the limbus intact.</li>
		<li>Place the cornea in a 60 mm dish with 1 mL of PBS, wound side down until mounting.</li>
	</ol>
	</li>
	<li>Mounting
	<ol>
		<li>Make sure the agar has come to a warm temperature (approximately 25 &#176;C).</li>
		<li>With two pairs of forceps, create a cup by holding two sides of the cornea with the endothelial side up. Add the warmed-up agar solution into the cornea using a sterile transfer pipette until it is full.</li>
		<li>After the agar hardens (usually about 30-45 s) carefully flip the cornea with the agar into 60 mm plate (Figure 3E). Cover with a lid.</li>
	</ol>
	</li>
	<li>Incubation
	<ol>
		<li>Add 4 mL of SSFM to the plate, maintaining corneas at an air-liquid interface at the limbal border in 5% CO2 at 37 &#176;C. Refresh media after 24 h and thereafter every other day. 
		NOTE: If performing a transfection into the wound, omit the antibiotics until after transfection.</li>
		<li>Wet the corneal surface once daily by adding 1 drop of SSFM from the conditioned media in the dish to maintain moisture. For this, take the dish out of the incubator, place it under the hood, remove the dish lid, wet the surface with media from dish using a sterile pipette, cover again and put it back at the incubator.</li>
		<li>For gene knockdown, treat the wound with gene-targeting or control siRNA that is complexed to a lipid-mediated carrier as per the supplier&#39;s instructions (see below).</li>
		<li>Mix 5 &#181;L (50 pmol) of siRNA into 50 &#181;L of reduced-serum minimum essential media (e.g., Opti-MEM). Mix 2 &#181;L of transfection reagent into 50 &#181;L of reduced-serum media. Let this sit for 5 min and then mix them.</li>
		<li>Add 200 &#181;L of reduced-serum minimum media to the reagent/siRNA mixture.</li>
		<li>Pipette dropwise onto the wound and incubate for 3 h.</li>
		<li>Wash out siRNA from corneal surface with media in the dish.Change the incubation media to SSFM + antibiotics (see the Table of Materials). Continue incubation as mentioned previously (steps 1.6.1-1.6.2).</li>
	</ol>
	</li>
</ol>
2. Histology: Paraffin Sections and Immunostaining
<ol>
	<li>Preparing the Tissue
	<ol>
		<li>After a two-week incubation, if using some of the tissue for quantitative real time polymerase chain reaction (qRT-PCR) analysis, before fixing, cut the cornea in half through the wound. Place this half or only &#188; (either is enough tissue) into stabilizing RNA-protect reagent.</li>
		<li>Using a standard isolation kit, isolate RNA and perform qRT-PCR. 
		NOTE: Alternatively, the wounded part only can be isolated and tested for gene expression.</li>
		<li>Place the other half of the cornea into tissue pathology cassettes and submerge in fixative (10% formalin) for 2-4 days at room temperature (RT).</li>
		<li>Paraffin embed this half of the wounded cornea using standard techniques. 
		NOTE: Orient the wounded cornea to ensure that the tissue sectioning will produce a cross-section of the cornea.</li>
	</ol>
	</li>
	<li>Immunostaining using 3,3&#39;-diaminobenzidine (DAB)
	<ol>
		<li>Day 1
		<ol>
			<li>Label slides properly using a pencil. De-paraffinize the tissue by placing slides into a jar with clearing agent (2 changes, 10 min each).</li>
			<li>Rehydrate the tissue by transferring the slides into ethanol at decreasing concentrations (100%, 100%, 70%, 50%, dH2O, dH20, 5 min for each change).</li>
			<li>Perform antigen retrieval by microwaving the slides in a plastic jar with citrate buffer (10 mM, pH 6.4) for 5 min. First cycle 5 min at 50% power. Refill the jar with citrate buffer and repeat. Cool down for 10 min.</li>
			<li>Wash 3x with PBS, 2 min each. Permeabilize tissue with 1% Triton X-100 in PBS 10 min at RT. Block sections with 3% normal goat serum (NGS) for 1 h at RT in humid chamber.</li>
			<li>Incubate tissue with primary antibody (1:100 or as the supplier suggests) in 3% NGS overnight at 4 &#176;C (300 &#181;L per slide).</li>
		</ol>
		</li>
		<li>Day 2
		<ol>
			<li>Rinse slides 3x with PBST (PBS plus 1% Tween 20), 2 min each. Place slides in 3% H2O2 for 10 min to block endogenous peroxidase. Wash 3x with PBST, 2 min each. Incubate sections with HRP secondary antibody (1:250) in 3% NGS for 1 h at RT (300 &#181;L per slide).</li>
			<li>Wash slides 3x PBST, 2 min each. Treat slides with the DAB kit. Add 300 &#181;L/slide for 3 min. Wash slides with dH2O 2x (quick dips).</li>
			<li>Counterstain with Hematoxylin for 20 s. Wash the slide with dH2O 2x (quick dips). Stain with bluing agent for 20 s. Rinse in dH2O for 20 s. Dehydrate tissue by placing slides into increasing concentrations of ethanol (50%, 70%, 100%, 100%, all quick dips).</li>
			<li>Dry slides on a paper towel under the hood for 10-20 min.</li>
			<li>Mount slides using 1 drop of mounting media, cover with coverslip. Label and store at RT.</li>
			<li>Image the slides under a microscope and quantify DAB signal with ImageJ7 (see section 4).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Immunostaining: Fluorescence
	<ol>
		<li>On Day 1 follow steps described in step 2.2.1. Perform the following steps on Day 2.</li>
		<li>Rinse slides 3x in PBST for 2 min each. Incubate sections with fluorophore-tagged secondary antibody in 3% NGS (1:200) for 1 h at RT.</li>
		<li>Wash 3x in PBST, 2 min each. Mount slides using 1 drop of 4&#8242;,6-diamidino-2-phenylindole (DAPI) mounting media and cover with a coverslip. Dry on paper towel under the hood for 30 min. Store in the dark at 4 &#176;C until fluorescence imaging.</li>
	</ol>
	</li>
	<li>Quantification using Image J
	<ol>
		<li>Download the &#34;Fiji&#34; version of ImageJ, which includes the necessary plugins for DAB staining quantification.</li>
		<li>Open an image in Fiji and select Image &#8594;&#160;Color &#8594;&#160;Color Deconvolution.</li>
		<li>Select &#34;H DAB&#34; as the stain and then click OK. Three new images will appear. Select the image that contains only DAB staining.</li>
		<li>To quantify stromal staining only, use the ImageJ eraser function to remove the epithelium from the DAB image.</li>
		<li>Select Analyze &#8594; Measure (or Ctrl + M) and record the value.</li>
	</ol>
	</li>
</ol>

The authors declare that they have no competing financial interests.

This protocol describes a model for studying wound healing in a natural stratified 3D environment. Use of organ culture as an intermediate between cell culture and in vivo studies significantly reduces costs as well as reducing procedures on live animals. Other 3D models have been of great benefit to the field including self-synthesizing collagen gels made from primary human corneal fibroblasts2 or these same cells embedded in gels made from animal-derived collagens31. The ...

Scarring in the cornea resulting from injury, trauma, or infection can lead to debilitating opacities and permanent vision loss. Thus, there is a critical need to identify pathways that can be targeted for therapeutic intervention. Current treatment options are limited and consist primarily of corneal transplantations, which are not accessible to patients across the world. Both human (Figure 1) and animal corneas can be utilized for 2D and 3D cell culture studies1,2. Human cadaver corneas not suitable for transplant can be obtained from eye banks or centralized tissue banks (National Disease Research Interchange (NDRI)), and animal eyes can be obtained from an abattoir. Primary corneal epithelial cells, stromal fibroblasts, and more recently, endothelial cells, can be isolated and cultured from these tissues for wound healing and toxicology studies3,4,5. In addition to the importance of understanding the molecular basis of blinding eye disease, the accessibility of tissue and the ability to culture primary cells has made the cornea an important model system for study. The cornea is ideal for testing the effects of agents on scarring as the normal cornea is transparent and certain types of wounds create opacities or fibrotic scars (reviewed in6). Several in vivo corneal wound healing models have also been extensively utilized for scarring studies1. Less utilized has been the ex vivo corneal wound healing model7,8 that is describe in detail here. The goal of this method is to quantify scarring outcomes characterized by fibrotic makers in a 3D multicellular corneal ex vivo model system.
Corneal epithelial wounding that does not breach the epithelial basement membrane normally closes within 24-72 h9. Soon after wounding, the cells at the edge of the epithelium start spreading and migrating into the epithelial free surface, to reestablish epithelial barrier function. This activity is sequentially followed by activation of corneal basal cell proliferation first and, in a later stage, of precursor cells located at the outer limbal zone to achieve recovery of epithelial cell mass10,11. These wounds often heal without scarring. However, a wound that penetrates the basement membrane into the stroma often results in scar formation1. After corneal stromal wounding, the stroma is populated with cells of multiple origins including differentiated resident stromal cells as well as bone marrow-derived fibrocytes12,13,14. Fibrotic scarring is characterized by the persistence of myofibroblasts in a healing wound. These pathological myofibroblasts demonstrate increased adhesion through the accumulation of integrins in focal adhesions, contractile &#945;-smooth muscle actin (&#945;-SMA) stress fibers, and local activation of extracellular matrix (ECM)-sequestered latent-transforming growth factor-beta (TGF&#946;). The differentiation of epithelial-derived cells, known as epithelial to mesenchymal transition (EMT), may also contribute to scar formation6.
There is a delicate balance between cell differentiation and apoptosis after wounding. Because of the breach in the basement membrane, growth factors such as platelet-derived growth factor (PDGF) and TGF&#946; from tears and the epithelium bathe the stroma, inducing myofibroblast differentiation, a sustained autocrine loop of TGF&#946;&#160;activation, and the secretion of disorganized fibrotic ECM15,16. The persistence of myofibroblasts in the healed wound promotes haze and scarring in the cornea (Figure 2). However, in regeneratively healed wound although myofibroblasts develop, they apoptose and thus are absent or significantly reduced in number in the healed tissue (reviewed in reference6,10). Thus, research on fibrotic scarring has focused at least in part on targeting molecules that prevent excessive myofibroblast development or myofibroblast persistence17,18. Because myofibroblast persistence characterizes both scarring and fibrotic disease in all tissues19, the cornea may be useful as a model system to study general cellular mechanisms of fibrosis.
In our model system, the cornea is wounded with a cylindrical blade called a trephine while still in the globe. Human and pig corneas can be wounded with either a 6 or 7 mm trephine; for rabbit corneas a 6 mm trephine is preferred. The pig cornea is similar in size to the human cornea. Because they are cost effective and readily available in large numbers, pig corneas are routinely used for organ culture. Furthermore, antibodies and siRNAs made to react with human have consistently cross-reacted with pig7. After wounding, corneas are cut from the globe with the limbus intact and mounted on an agar/collagen base. The corneas are cultured in serum-free media plus stabilized vitamin C to simulate fibroblast proliferation and ECM deposition20. Neither the addition of serum nor growth factors are needed to induce myofibroblast formation7. Corneas are routinely fixed and processed for histology after two weeks of culture. For gene knockdown, or to test the effects of an agent on wound healing, the wound can be treated with siRNA in the wound after wounding7 or a soluble agent can be added to the media, respectively8.

<table><tbody><tr><td>PBS</td><td>Gibco</td><td>10-010-023</td><td></td></tr><tr><td>Pen Strep&amp;nbsp;</td><td>MP Biomedicals</td><td>91670049</td><td></td></tr><tr><td>Bovine Collagen Solution</td><td>Advance Biomatrix</td><td>5005</td><td></td></tr><tr><td>Pig eyes with lids attached&amp;nbsp;</td><td>Pel-freeze, Arkansas</td><td>N/A</td><td></td></tr><tr><td>6.0 mm trephine&amp;nbsp;</td><td>Katena</td><td>K28014</td><td></td></tr><tr><td>Surgical Blade&amp;nbsp;</td><td>Personna</td><td>0.009</td><td></td></tr><tr><td>Small scissor</td><td>Fisher</td><td>895110</td><td></td></tr><tr><td>Forceps</td><td>Fisher</td><td>08953-F</td><td></td></tr><tr><td>Kim Wipes&amp;nbsp;</td><td>Kimberly-Clark&amp;trade; 34120</td><td>06-666</td><td></td></tr><tr><td>60 mm cell culture dishes&amp;nbsp;</td><td>Falcon</td><td>08-772B</td><td></td></tr><tr><td>&lt;strong&gt;Supplemented Serum- Free media (SSFM)&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;Add all of the following components to DMEM/F-12:&amp;nbsp; ITS, RPMI, Glutathione, L-Glutamine, MEM Non essential amino acids, MEM Sodium Pyruvate, ABAM, Gentamicin, Vitamin C.&amp;nbsp;&lt;/strong&gt;</td></tr><tr><td>DMEM/F-12</td><td>Gibco</td><td>11330</td><td></td></tr><tr><td>ITS Liquid Media Supplement&amp;nbsp;</td><td>Sigma</td><td>I3146</td><td>100X</td></tr><tr><td>RPMI 1640 Vitamins Solution&amp;nbsp;</td><td>Sigma</td><td>R7256</td><td>100x</td></tr><tr><td>Glutathione</td><td>Sigma</td><td>G6013</td><td>Use at 1 &amp;micro;g/mL. Freeze aliquots; do not reuse after thawing.</td></tr><tr><td>1% L-glutamine solution&amp;nbsp;</td><td>Gibco</td><td>25030-081</td><td>100x</td></tr><tr><td>MEM Non-essential amino acids solution&amp;nbsp;</td><td>Gibco</td><td>11140</td><td>100x</td></tr><tr><td>MEM Sodium pyruvate solution&amp;nbsp;</td><td>Gibco</td><td>11360</td><td>1 M Stocks (1,000x) and freeze in single use aliquits.&amp;nbsp; Use from freezer each time media is made.</td></tr><tr><td>ABAM&amp;nbsp;</td><td>Sigma</td><td>A7292</td><td>100x</td></tr><tr><td>Gentamicin&amp;nbsp;</td><td>Sigma</td><td>30-005-CR</td><td>200x</td></tr><tr><td>Vitamin C&amp;nbsp;</td><td>Wako</td><td>070-0483</td><td>2-0-aD Glucopyranosyl-Ascorbic Acid. 1 mM stocks (1000x)</td></tr><tr><td>10% Iodine&amp;nbsp;</td><td>Fisher Chemical</td><td>SI86-1</td><td></td></tr><tr><td>Tissue Path Cassettes&amp;nbsp;</td><td>Fisher</td><td>22-272416</td><td></td></tr><tr><td>Normal Goat Serum (NGS)</td><td>Jackson Immuno Research</td><td>005-000-121</td><td>We use 3% NGS</td></tr><tr><td>Mounting Media&amp;nbsp;</td><td>Thermo Scientific</td><td>TA-030-FM</td><td></td></tr><tr><td>Safe Clear&amp;nbsp;</td><td>Fisher</td><td>314-629</td><td></td></tr><tr><td>Ethyl Alcohol</td><td>Ultra Pure</td><td>200CSGP</td><td>200 Proof, diluted at 100%, 70%, 50%)&amp;nbsp;</td></tr><tr><td>Sodium citrate&amp;nbsp;</td><td>Fisher</td><td>BP327</td><td>10 mM, pH 6.4</td></tr><tr><td>Hematoxylin</td><td>EMD Millipore</td><td>M10742500</td><td></td></tr><tr><td>Bluing agent&amp;nbsp;</td><td>Ricca Chemical Company</td><td>220-106</td><td></td></tr><tr><td>1% Triton X-100</td><td>Fisher</td><td>9002-93-1</td><td>Diluted in PBS</td></tr><tr><td>0.1% Tween 20&amp;nbsp;</td><td>Fisher</td><td>BP337</td><td>Diluted in PBS</td></tr><tr><td>3% Hydrogen Peroxide&amp;nbsp;</td><td>Fisher</td><td>H324</td><td></td></tr><tr><td>DAB Kit&amp;nbsp;</td><td>Vector Laboratories</td><td>SK-4100</td><td></td></tr><tr><td>Agar&amp;nbsp;</td><td>Fisher&amp;nbsp;</td><td>BP1423-500</td><td>Agar solution: prepare 1% agar and 1 mg/mL bovine collagen in DMEM-F12 up to 20 mL</td></tr><tr><td>Parafilm</td><td>Bermis</td><td>13-374-12</td><td></td></tr><tr><td>Moist Chamber</td><td></td><td></td><td>Use any chamber, cover it with wet Wipe Tissue and then put a layer of Parafilm over it.</td></tr><tr><td>Lipofectamine 2000</td><td></td><td></td><td></td></tr><tr><td>Qiagen RNAprotect Cell Reagent</td><td>Qiagen</td><td>&amp;nbsp;76104</td><td></td></tr><tr><td>Ambion PureLink RNA Mini Kit</td><td>Thermo Scientific</td><td>12183018A</td><td></td></tr><tr><td>Anti-Fibronectin-EDA Antibody</td><td>Sigma</td><td>F6140</td><td>1:200 Diluted in&amp;nbsp; 3% normal goat serum</td></tr><tr><td>Anti-alpha smooth muscle actin Antibody</td><td>Sigma</td><td>A2547 or C6198 (cy3 conjugated)</td><td>1:200 Diluted in 3% normal goat serum</td></tr><tr><td>Permafluor&amp;nbsp;</td><td>Thermo Scientific</td><td>TA-030-FM</td><td></td></tr><tr><td>DAPI&amp;nbsp;</td><td>Invitrogen</td><td>P36931</td><td></td></tr><tr><td>Gt anti -MS IgG (H+L) Secondary Antibody, HRP&amp;nbsp;</td><td>Invitrogen</td><td>62-6520</td><td>1:100 diluted in 3% normal goat serum (for a-SMA, DAB staining)</td></tr><tr><td>Gt anti -MS IgM (H+L) Secondary Antibody, HRP&amp;nbsp;</td><td>Thermo Scientific</td><td>PA1-85999</td><td>1:100 diluted in 3% normal goat serum (for FN-EDA, DAB staining)</td></tr><tr><td>Gt anti -MS IgG (H+L) Secondary Antibody, Cy3&amp;nbsp;</td><td>Jackson Immuno Research</td><td>115-165-146</td><td>1:200 Diluted in&amp;nbsp; 3% normal goat serum (for a-SMA, Fluorescence staining)</td></tr><tr><td>&amp;nbsp;Zeiss Axioplan2&amp;nbsp;</td><td>Zeiss</td><td></td><td>Microscope</td></tr><tr><td>SPOT-2</td><td>Diagnostic Instruments, Sterling Heights, Michigan</td><td>CCD camera</td><td></td></tr></tbody></table>

ex vivo corneal organ culture model for wound healing studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

Immunohistochemistry is the primary assay utilized to analyze the success of the ex vivo wound healing experiment. Figure 4 depicts the epithelium and anterior stroma in control tissue (Figure 4A, 4B). Six hours after wounding, the epithelium was absent (Figure 4C, 4D). Six days after wounding as expected, the epithelium had regrown (Figure 4E</s...

Watch this Scientific Journal Video about Ex Vivo Corneal Organ Culture Model for Wound Healing Studies at JoVE.com

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

ex-vivo-corneal-organ-culture-model-for-wound-healing-studies

Research

JoVE Journal

Medicine

11.6K Views.  SUNY Upstate Medical University. Dit document beschreef een eenvoudige methode voor het testen van wond in een driedimensionaal, multicellulair orgaancultuurmodelsysteem. We doen dit met behulp van varkensogen, maar zelfs als je niet bekend bent met de ogen, u deze test doen. Als overzicht kregen we varkensogen, knipten de bollen uit, maken een cirkelvormige wond in het hoornvlies dat door het epitheel gaat en ongeveer een derde van de stroma.We snijden het hoornvlies uit, en monteren het op een Agar basis, en zetten ...

Video: Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

Developmental Biology

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as Aniridia or Steven Johnson Syndrome as well as by external factors such as chemical burns or radiation. Current treatments are (i) the use of corneal grafts and (ii) the use of stem cell expanded in the laboratory and delivered on carriers (e.g., amniotic membrane); these treatments are relatively successful but unfortunately they can fail after 3-5 years. There is a need to design and manufacture new corneal biomaterial devices able to mimic in detail the physiological environment where stem cells reside in the cornea. Limbal stem cells are located in the limbus (circular area between cornea and sclera) in specific niches known as the Palisades of Vogt. In this work we have developed a new platform technology which combines two cutting-edge manufacturing techniques (microstereolithography and electrospinning) for the fabrication of corneal membranes that mimic to a certain extent the limbus. Our membranes contain artificial micropockets which aim to provide cells with protection as the Palisades of Vogt do in the eye.

We report a technique for the fabrication of micropockets within electrospun membranes in which to study cell behavior. Specifically, we describe a combination of microstereolithography and electrospinning for the production of PLGA (Poly(lactide-co-glycolide)) corneal biomaterial devices equipped with microfeatures.

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as ...

Bioengineering

Investigating Scarless Tissue Regeneration in Embryonic Wounded Chick Corneas

Chick embryonic corneal wounds display a remarkable capacity to fully and rapidly regenerate, whereas adult wounded corneas experience a loss of transparency due to fibrotic scarring. The tissue integrity of injured embryonic corneas is intrinsically restored with no detectable scar formation. Given its accessibility and ease of manipulation, the chick embryo is an ideal model for studying scarless corneal wound repair. This protocol demonstrates the different steps involved in wounding the cornea of an embryonic chick in ovo. First, eggs are windowed at early embryonic ages to access the eye. Second, a series of in ovo physical manipulations to the extraembryonic membranes are conducted to ensure access to the eye is maintained through later stages of development, corresponding to when the three cellular layers of the cornea are formed. Third, linear cornea wounds that penetrate the outer epithelial layer and the anterior stroma are made using a microsurgical knife. The regeneration process or fully restored corneas can be analyzed for regenerative potential using various cellular and molecular techniques following the wounding procedure. Studies to date using this model have revealed that wounded embryonic corneas display activation of keratocyte differentiation, undergo coordinated remodeling of ECM proteins to their native three-dimensional macrostructure, and become adequately re-innervated by corneal sensory nerves. In the future, the potential impact of endogenous or exogenous factors on the regenerative process could be analyzed in healing corneas by using developmental biology techniques, such as tissue grafting, electroporation, retroviral infection, or bead implantation. The current strategy identifies the embryonic chick as a crucial experimental paradigm for elucidating the molecular and cellular factors coordinating scarless corneal wound healing.

The present protocol demonstrates the different steps involved in wounding the cornea of an embryonic chick in ovo. The regenerating or fully restored corneas can be analyzed for regenerative potential using various cellular and molecular techniques following the wounding procedure.&#160;

Chick embryonic corneal wounds display a remarkable capacity to fully and rapidly regenerate, whereas adult wounded corneas experience a loss of ...

Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to animal infections in vivo. The existing in vitro tests typically overestimate antimicrobial efficacy as the presence of host tissue as a diffusion barrier is not accounted for. To overcome this bottleneck, we have developed an ex vivo porcine corneal model of bacterial keratitis using Pseudomonas aeruginosa as a prototypic organism. This article describes the preparation of the porcine cornea and protocol for establishment of the infection. Bespoke glass molds enable straightforward setup of the cornea for infection studies. The model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue. Establishment of infection is verified as an increase in the number of colony forming units assessed via viable plate counts. The results demonstrate that infection can be established in a highly reproducible fashion in the ex vivo corneas using the method described here. The model can be extended in the future to mimic keratitis caused by microorganisms other than P. aeruginosa. The ultimate aim of the model is to investigate the effect of antimicrobial chemotherapy on the progress of bacterial infection in a scenario more representative of in vivo infections. In so doing, the model described here will reduce the use of animals for testing, improve success rates in clinical trials and ultimately enable rapid translation of novel antimicrobials to the clinic.

This article describes a step-by-step protocol to set up an ex vivo porcine model of bacterial keratitis. Pseudomonas aeruginosa is used as a prototypic organism. This innovative model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue.

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to ...

Immunology and Infection

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

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 ...

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...

An Epithelial Abrasion Model for Studying Corneal Wound Healing

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its transparency. However, due to its external position, the cornea is highly susceptible to a wide variety of injuries that can lead to the loss of corneal transparency and eventual blindness. Efficient corneal wound healing in response to these injuries is pivotal for maintaining corneal homeostasis and preservation of corneal transparency and refractive capabilities. In events of compromised corneal wound healing, the cornea becomes vulnerable to infections, ulcerations, and scarring. Given the fundamental importance of corneal wound healing to the preservation of corneal transparency and vision, a better understanding of the normal corneal wound healing process is a prerequisite to understanding impaired corneal wound healing associated with infection and disease. Toward this goal, murine models of corneal wounding have proven useful in furthering our understanding of the corneal wound healing mechanisms operating under normal physiological conditions. Here, a protocol for creating a central corneal epithelial abrasion in mouse using a trephine and a blunt golf club spud is described. In this model, a 2 mm diameter circular trephine, centered over the cornea, is used to demarcate the wound area. The golf club spud is used with care to debride the epithelium and create a circular wound without damaging the corneal epithelial basement membrane. The resulting inflammatory response proceeds as a well-characterized cascade of cellular and molecular events that are critical for efficient wound healing. This simple corneal wound healing model is highly reproducible and well-published and is now being used to evaluate compromised corneal wound healing in the context of disease.

Here, a protocol for creating a central corneal epithelial abrasion wound in the mouse using a trephine and a blunt golf club spud is described. This corneal wound healing model is highly reproducible and is now being used to evaluate compromised corneal wound healing in the context of diseases.

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its ...

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Here, animal models based on mouse and rabbit are developed for mechanical and chemical injury of corneal epithelium to screen new therapeutics and the underlying mechanism.

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, ...

A Human Corneal Organ Culture Model of Descemet's Stripping Only with Accelerated Healing Stimulated by Engineered Fibroblast Growth Factor 1

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the whole cornea or Descemet's membrane. Recent developments in ocular surgery have established Descemet's Stripping Only (DSO), a surgical technique in which a central circle of guttae-dense Descemet's membrane is removed to allow for the migration of CECs onto the smooth stroma, restoring function and vision to the cornea. While this potential treatment option is of high interest in the field of ophthalmic research, no successful ex vivo models of DSO have been established and clinical data is limited. This work presents a novel wound-healing model simulating DSO in human donor corneas. Using this approach to evaluate the efficacy of the human engineered FGF1 (NM141), we found that treatment accelerated healing via stimulation of migration and proliferation of CECs. This finding was confirmed in 11 pairs of human corneas with signs of dystrophy reported by the eye banks in order to verify that these results can be replicated in patients with Fuchs' Dystrophy, as the target population of the DSO procedure.

Descemet's Stripping Only is an experimental procedure wherein patients with central corneal guttae resulting from Fuchs Endothelial Corneal Dystrophy have Descemet's membrane stripped for peripheral cells to regenerate the endothelial layer. We present novel methodology simulating DSO in dystrophic human corneas ex vivo with accelerated healing stimulated by eFGF1 (NM141).

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

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