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

Summary

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.

Abstract

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’ 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.

Introduction

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 woun....

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Protocol

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 Vivo Wound Culture

1. 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, 8.25 mM Tris Base, pH to 7.4 with HCl) at RT, leaving the third empty. Pre-warm culture media (Media 199 supplemented with, 1% L-glutamine and 1% peni....

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Results

Ex Vivo Model created to study the wound healing process in the cells’ native microenvironment

To investigate mechanisms involved in regulating wound healing of an epithelium within the cells’ 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 .......

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Discussion

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 tissues^2,4. While the protocol is straightforward and simple to follow, performing mock cataract surgery with embryoni.......

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Materials

Name	Company	Catalog Number	Comments
Sodium Chloride (NaCl)	Fisher Scientific	S271-3	Use at 140 mM in TD Buffer
Potassium Chloride (KCl)	Fisher Scientific	P217-500	Use at 5 mM in TD Buffer
Sodium Phosphate (Na2HPO4)	Sigma	S0876	Use at 0.7 mM in TD Buffer
D-glucose (Dextrose)	Fisher Scientific	D16-500	Use at 0.5 mM in TD Buffer
Tris Base	Fisher Scientific	BP152-1	Use at 8.25 mM in TD Buffer
Hydrochloric acid	Fisher Scientific	A144-500	Use to pH TD buffer to 7.4
Media 199	GIBCO	11150-059
L-glutamine	Corning/CellGro	25-005-CI	Use at 1% in Media199
Penicillin/streptomycin	Corning/CellGro	30-002-CI	Use at 1% in Media199
100 mm petri dishes	Fisher Scientific	FB0875711Z
Stericup Filter Unit	Millipore	SCGPU01RE	Use to filter sterilize Media
Dumont #5 forceps (need 2)	Fine Science Tools	11251-20
35 mm Cell Culture Dish	Corning	430165
27 G 1 ml SlipTip with precision glide needle	BD	309623
Fine Scissors	Fine Science Tools	14058-11
Standard Forceps	Fine Science Tools	91100-12
Other Items Needed: General dissection instruments,  fertile white leghorn chicken eggs, check egg incubator (humidified, 37.7°C), laminar flow hood, binocular stereovision dissecting microscope