This work was supported by National Institutes of Health Grant to A.S.M. (EY021784).

The following protocol complies with the Thomas Jefferson University Institutional Animal Care and Use Committee guidelines and with the ARVO Statement for the Use of Animals in Vision Research.
1. Setup and Preparation of Lenses for Ex&#160;Vivo Wound Culture
<ol>
	<li>Place three 100 mm petri dishes in a sterile, laminar flow hood. Fill two of the petri dishes halfway with Tris/Dextrose buffer (TD buffer; 140 mM NaCl, 5 mM KCl, 0.7 mM Na2PO4, 5 mM D-glucose, ...

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+wound-healing+assays+for+probing+collective+cell+migration.">Advances in wound-healing assays for probing collective cell migration.</a> Journal of laboratory automation. 17, 59-65 (2012).</li><li>Khalil, A. A., Friedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+leader+cells+in+collective+cell+migration.">Determinants of leader cells in collective cell migration.</a> Integrative biology : quantitative biosciences from nano to macro. 2, 568-574 (2010).</li><li>Walker, J. L., Wolff, I. M., Zhang, L., Menko, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+SRC+kinases+signals+induction+of+posterior+capsule+opacification.">Activation of SRC kinases signals induction of posterior capsule opacification.</a> Investigative ophthalmology &amp; visual science. 48, 2214-2223 (2007).</li><li>Sta Iglesia, D. D., Stepp, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+the+basement+membrane+after+corneal+debridement.">Disruption of the basement membrane after corneal debridement.</a> Investigative ophthalmology &amp; visual science. 41, 1045-1053 (2000).</li><li>Pal-Ghosh, S., Pajoohesh-Ganji, A., Brown, M., Stepp, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+for+the+study+of+recurrent+corneal+epithelial+erosions:+alpha9beta1+integrin+implicated+in+progression+of+the+disease.">A mouse model for the study of recurrent corneal epithelial erosions: alpha9beta1 integrin implicated in progression of the disease.</a> Investigative ophthalmology &amp; visual science. 45, 1775-1788 (2004).</li><li>Pal-Ghosh, S., Pajoohesh-Ganji, A., Tadvalkar, G., Stepp, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+the+basement+membrane+enhances+corneal+wound+healing.">Removal of the basement membrane enhances corneal wound healing.</a> Experimental eye research. 93, 927-936 (2011).</li><li>Stepp, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wounding+the+cornea+to+learn+how+it+heals.">Wounding the cornea to learn how it heals.</a> Experimental eye research. 121, 178-193 (2014).</li><li>Kuwabara, T., Perkins, D. G., Cogan, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sliding+of+the+epithelium+in+experimental+corneal+wounds.">Sliding of the epithelium in experimental corneal wounds.</a> Investigative ophthalmology. 15, 4-14 (1976).</li><li>Sherrard, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+corneal+endothelium+in+vivo:+its+response+to+mild+trauma.">The corneal endothelium in vivo: its response to mild trauma.</a> Experimental eye research. 22, 347-357 (1976).</li><li>Stramer, B. M., Zieske, J. D., Jung, J. C., Austin, J. S., Fini, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+controlling+the+fibrotic+repair+phenotype+in+cornea:+implications+for+surgical+outcomes.">Molecular mechanisms controlling the fibrotic repair phenotype in cornea: implications for surgical outcomes.</a> Investigative ophthalmology &amp; visual science. 44, 4237-4246 (2003).</li><li>Escamez, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vivo+model+of+wound+healing+in+genetically+modified+skin-humanized+mice.">An in vivo model of wound healing in genetically modified skin-humanized mice.</a> The Journal of investigative dermatology. 123, 1182-1191 (2004).</li><li>Werner, S., Breeden, M., Hubner, G., Greenhalgh, D. G., Longaker, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+keratinocyte+growth+factor+expression+is+reduced+and+delayed+during+wound+healing+in+the+genetically+diabetic+mouse.">Induction of keratinocyte growth factor expression is reduced and delayed during wound healing in the genetically diabetic mouse.</a> The Journal of investigative dermatology. 103, 469-473 (1994).</li><li>Tarin, D., Croft, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+studies+of+wound+healing+in+mouse+skin.+II.+Dermo-epidermal+interrelationships.">Ultrastructural studies of wound healing in mouse skin. II. Dermo-epidermal interrelationships.</a> Journal of anatomy. 106, 79-91 (1970).</li><li>Croft, C. B., Tarin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+studies+of+wound+healing+in+mouse+skin+I.+Epithelial+behaviour.">Ultrastructural studies of wound healing in mouse skin I. Epithelial behaviour.</a> Journal of anatomy. 106, 63-77 (1970).</li><li>Winstanley, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epithelial+reaction+in+the+healing+of+excised+cutaneous+wounds+in+the+dog.">The epithelial reaction in the healing of excised cutaneous wounds in the dog.</a> Journal of comparative pathology. 85, 61-75 (1975).</li><li>Wormstone, I. M., Wride, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ocular+lens:+a+classic+model+for+development,+physiology+and+disease.">The ocular lens: a classic model for development, physiology and disease.</a> Philosophical transactions of the Royal Society of London. Series B, Biological. 366, 1190-1192 (2011).</li><li>Danysh, B. P., Duncan, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lens+capsule.">The lens capsule.</a> Experimental eye research. 88, 151-164 (2009).</li><li>Awasthi, N., Guo, S., Wagner, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posterior+capsular+opacification:+a+problem+reduced+but+not+yet+eradicated.">Posterior capsular opacification: a problem reduced but not yet eradicated.</a> Archives of ophthalmology. 127, 555-562 (2009).</li><li>Walker, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+attempts+to+reduce+posterior+capsule+opacification+after+cataract+surgery--a+review.">Pharmacological attempts to reduce posterior capsule opacification after cataract surgery--a review.</a> Clinical &amp; experimental ophthalmology. 36, 883-890 (2008).</li><li>Schmidbauer, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posterior+capsule+opacification.">Posterior capsule opacification.</a> International ophthalmology clinics. 41, 109-131 (2001).</li><li>Menko, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+central+role+for+vimentin+in+regulating+repair+function+during+healing+of+the+lens+epithelium.">A central role for vimentin in regulating repair function during healing of the lens epithelium.</a> Molecular biology of the cell. 25, 776-790 (2014).</li><li>Chauss, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+state-specific+mitochondrial+dynamic+regulatory+networks+are+revealed+by+global+transcriptional+analysis+of+the+developing+chicken+lens.">Differentiation state-specific mitochondrial dynamic regulatory networks are revealed by global transcriptional analysis of the developing chicken lens.</a> G3 (Bethesda). 4, 1515-1527 (2014).</li><li>Leonard, M., Zhang, L., Bleaken, B. M., Menko, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+roles+for+N-Cadherin+linked+c-Src+and+fyn+kinases+in+lens+development.">Distinct roles for N-Cadherin linked c-Src and fyn kinases in lens development.</a> Developmental dynamics : an official publication of the American Association of Anatomists. 242, 469-484 (2013).</li><li>Sieg, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FAK+integrates+growth-factor+and+integrin+signals+to+promote+cell+migration.">FAK integrates growth-factor and integrin signals to promote cell migration.</a> Nature cell biology. 2, 249-256 (2000).</li><li>Sieg, D. J., Hauck, C. R., Schlaepfer, D. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional-specific+alterations+in+cell-cell+junctions,+cytoskeletal+networks+and+myosin-mediated+mechanical+cues+coordinate+collectivity+of+movement+of+epithelial+cells+in+response+to+injury.">Regional-specific alterations in cell-cell junctions, cytoskeletal networks and myosin-mediated mechanical cues coordinate collectivity of movement of epithelial cells in response to injury.</a> Experimental cell research. 322, 133-148 (2014).</li><li>Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing--aiming+for+perfect+skin+regeneration.">Wound healing--aiming for perfect skin regeneration.</a> Science. 276, 75-81 (1997).</li><li>Ferguson, M. W., O'Kane, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scar-free+healing:+from+embryonic+mechanisms+to+adult+therapeutic+intervention.">Scar-free healing: from embryonic mechanisms to adult therapeutic intervention.</a> Philosophical transactions of the Royal Society of London. Series B, Biological. 359, 839-850 (2004).</li><li>Redd, M. J., Cooper, L., Wood, W., Stramer, B., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+and+inflammation:+embryos+reveal+the+way+to+perfect+repair.">Wound healing and inflammation: embryos reveal the way to perfect repair.</a> Philosophical transactions of the Royal Society of London. 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Here is described a technique for preparing a culture model of wound repair that involves performing an ex vivo cataract surgery on chick embryo lenses after their removal from the eye. The lens epithelium responds to this clinically relevant wounding with a repair process that closely mimics that which occurs in vivo, and shares features with wound repair in other epithelial tissues2,4. While the protocol is straightforward and simple to follow, performing mock cataract surgery with embryoni...

The clinically relevant, mock cataract surgery, ex vivo epithelial wound healing model described here was developed to provide a tool for investigating the mechanisms that regulate repair of epithelial tissues in response to an injury. Key features that were aimed for in creating this model included 1) providing conditions that closely replicated the in vivo response to wounding in a culture setting, 2) ease of modulating the regulatory elements of repair, and 3) ability to image the repair process, in its entirety, in real time. The challenge, therefore, was to create a culture model in which it was possible to study, and manipulate, epithelial wound repair in the cells&#8217; native microenvironment. The availability of this wound-repair model opens new possibilities for identifying the endogenous signaling cues from matrix proteins, cytokines and chemokines that regulate the repair process. In addition, the model is ideal for examining how an epithelium is able to move as a collective sheet to re-epithelialize the wound area 2,3, and for determining the lineage of mesenchymal leader cells at the wound edge that function in directing the collective migration of the injured epithelium 4. This model also provides a platform with which to identify therapeutics that could promote effective wound healing and prevent aberrant wound repair5.
There are already a number of available wound-repair models, both in culture and in vivo, which have provided most of what is known about the wound repair process today. In animal injury models, such as cornea 6-12 and skin13-17, there is the opportunity to study the response of the tissue to wounding in the context of all the repair mediators that could be involved in the process, including contributions from the vasculature and nervous system. However, there are limitations to manipulating the experimental conditions in vivo, and it is not yet possible to conduct imaging studies of the repair response in vivo, continuously over time. In contrast, most in vitro wound-repair culture models, such as the scratch wound, can be easily manipulated and followed over time but lack the environmental context of studying wound healing in the in vivo tissue. While ex vivo models offer the advantage of studying the injury repair process continuously over time in the context of the cells&#8217; microenvironment coupled with the ability to modulate the molecular regulators of repair at any time point in the process, there are few models that fit these parameters.
Here is described a procedure to generate highly reproducible ex vivo epithelial wound healing cultures that reproduce an epithelial tissue&#8217;s response to a physiological wounding. Using the chick embryo lens as a tissue source, an ex vivo mock cataract surgery is performed. The lens is an ideal tissue to use for these studies since it is self-contained within a thick basement membrane capsule, avascular, not innervated, and free of any associated stroma18,19. In the human disease, cataract surgery addresses vision loss due to opacification of the lens, and involves removal of the lens fiber cell mass, which comprises the bulk of the lens. Following cataract surgery vision is restored through the insertion of an artificial intraocular lens. The cataract surgery procedure, through removal of fiber cells, induces an injury response in the adjacent lens epithelium, which responds by re-epithelialization of the posterior area of the lens capsule that had been occupied by the fiber cells. In cataract surgery, as in most wound repair responses, there sometimes occurs an aberrant fibrotic outcome to the wound healing response, associated with the emergence of myofibroblasts, which in the lens is known as Posterior Capsule Opacification20-22. To generate the cataract surgery wound healing model, a cataract surgery procedure is mimicked in lenses removed from the chick embryo eye to produce a physiological injury. Microsurgical removal of lens fiber cells results in a very consistent circular wound area surrounded by the lens epithelial cells. This cell population remains firmly attached to the lens basement membrane capsule and is injured by the surgical procedure. The epithelial cells migrate onto the denuded area of the endogenous basement membrane to heal the wound, led by a population of vimentin-rich mesenchymal cells known in the repair process as leader cells1. With this model the response of an epithelium to injury can be easily visualized and followed with time in the context of the cells&#8217; microenvironment. The cells are readily accessible to modifications of the expression or activation of molecules expected to play a role in wound repair. A powerful feature of this model is the ability to isolate and study migration-specific changes in the framework of wound healing. The ability to prepare large numbers of aged matched ex vivo wound healing cultures for studies is another advantage of this model. Thus, this model system provides a unique opportunity to tease apart mechanisms of wound repair and test therapeutics for their effect on the wound healing process. The ex vivo mock cataract surgery model is expected to have wide applicability, providing a critical resource for studying mechanisms of injury repair.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td>Use at 140mM in TD Buffer</td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td>Use at 5mM in TD Buffer</td>
		</tr>
		<tr>
			<td>Sodium Phosphate (Na2HPO4)</td>
			<td>Sigma</td>
			<td>S0876</td>
			<td>Use at .7mM in TD Buffer</td>
		</tr>
		<tr>
			<td>D-glucose (Dextrose)</td>
			<td>Fisher Scientific</td>
			<td>D16-500</td>
			<td>Use at 0.5mM in TD Buffer</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP152-1</td>
			<td>Use at 8.25mM in TD Buffer</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Fisher Scientific</td>
			<td>A144-500</td>
			<td>Use to pH TD buffer to 7.4</td>
		</tr>
		<tr>
			<td>Media 199</td>
			<td>GIBCO</td>
			<td>11150-059</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Corning/CellGro</td>
			<td>25-005-CI</td>
			<td>Use at 1% in Media199</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Corning/CellGro</td>
			<td>30-002-CI</td>
			<td>Use at 1% in Media199</td>
		</tr>
		<tr>
			<td>100mm petri dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875711Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup Filter Unit</td>
			<td>Millipore</td>
			<td>SCGPU01RE</td>
			<td>Use to filter sterilize Media</td>
		</tr>
		<tr>
			<td>Dumont #5 forceps (need 2)</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>35mm Cell Culture Dish</td>
			<td>Corning</td>
			<td>430165</td>
			<td></td>
		</tr>
		<tr>
			<td>27 Gauge 1mL SlipTip with precision glide needle</td>
			<td>BD</td>
			<td>309623</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>14058-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Forceps</td>
			<td>Fine Science Tools</td>
			<td>91100-12</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Other Items Needed: General dissection instruments,&#160; fertile white leghorn chicken eggs,&#160;</td>
		</tr>
		<tr>
			<td colspan="4">check egg incubator (humidified, 37.7&#176;C), laminar flow hood, binocular stereovision dissecting&#160;</td>
		</tr>
		<tr>
			<td>microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Ex Vivo Model created to study the wound healing process in the cells&#8217; native microenvironment
To investigate mechanisms involved in regulating wound healing of an epithelium within the cells&#8217; native microenvironment, a clinically relevant ex vivo mock cataract surgery model was created. This model is created from lens tissue which offers many advantages due to its intrinsic properties: 1) the lens is a self-contained organ surrounded by a thick basement membrane ...

Watch this Scientific Journal Video about Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment at JoVE.com

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.

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 ...

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JoVE Journal

Developmental Biology

6.5K 조회수.  Thomas Jefferson University. 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. 