Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

Summary

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.

Abstract

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.

Introduction

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 studies^1,2. Human cadaver corneas not suitable for transplant can be obtained from eye banks or centralized tissue banks (National Disease R....

Protocol

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 amount of SSFM to be prepared depends on the number of corneas to be processed. Usually 30 mL is enough media for 4 corneas.

Results

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

Discussion

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 .......

Materials

List of materials used in this article

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 (1,000x) 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	10 mM, 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