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Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

Published: February 15th, 2019



1Department of Ophthalmology, SUNY Upstate Medical University, 2Department of Dermatology, Columbia University Medical Center
* These authors contributed equally

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 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.

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 R....

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1. Organ Culture

  1. Preparations
    1. 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.
    2. Prepare supplemented serum-free media (SSFM) as per the composition provided in the Table of Materials.
      NOTE: The necessary am.......

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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

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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 .......

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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.


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

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