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 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 studies^3,4,5. 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 model^7,8 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 mass^10,11. 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 fibrocytes^12,13,14. 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 α-smooth muscle actin (α-SMA) stress fibers, and local activation of extracellular matrix (ECM)-sequestered latent-transforming growth factor-beta (TGFβ). 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 TGFβ from tears and the epithelium bathe the stroma, inducing myofibroblast differentiation, a sustained autocrine loop of TGFβ activation, and the secretion of disorganized fibrotic ECM^15,16. 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 reference^6,10). Thus, research on fibrotic scarring has focused at least in part on targeting molecules that prevent excessive myofibroblast development or myofibroblast persistence^17,18. 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.

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.
2. 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.
   1. 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).
   2. 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).
   3. 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.
3. Wounding
   1. 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.
   2. Place the trephine in the center of the cornea, rotate it 180° 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.
   3. 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).
4. Cutting and Removing the Cornea from the Globe
   1. 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.
   2. 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.
   3. Place the cornea in a 60 mm dish with 1 mL of PBS, wound side down until mounting.
5. Mounting
   1. Make sure the agar has come to a warm temperature (approximately 25 °C).
   2. 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.
   3. 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.
6. Incubation
   1. Add 4 mL of SSFM to the plate, maintaining corneas at an air-liquid interface at the limbal border in 5% CO₂ at 37 °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.
   2. 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.
   3. For gene knockdown, treat the wound with gene-targeting or control siRNA that is complexed to a lipid-mediated carrier as per the supplier's instructions (see below).
   4. Mix 5 µL (50 pmol) of siRNA into 50 µL of reduced-serum minimum essential media (e.g., Opti-MEM). Mix 2 µL of transfection reagent into 50 µL of reduced-serum media. Let this sit for 5 min and then mix them.
   5. Add 200 µL of reduced-serum minimum media to the reagent/siRNA mixture.
   6. Pipette dropwise onto the wound and incubate for 3 h.
   7. 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).

2. Histology: Paraffin Sections and Immunostaining

1. Preparing the Tissue
   1. 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 ¼ (either is enough tissue) into stabilizing RNA-protect reagent.
   2. 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.
   3. 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).
   4. 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.
2. Immunostaining using 3,3'-diaminobenzidine (DAB)
   1. Day 1
      1. Label slides properly using a pencil. De-paraffinize the tissue by placing slides into a jar with clearing agent (2 changes, 10 min each).
      2. Rehydrate the tissue by transferring the slides into ethanol at decreasing concentrations (100%, 100%, 70%, 50%, dH₂O, dH₂0, 5 min for each change).
      3. 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.
      4. 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.
      5. Incubate tissue with primary antibody (1:100 or as the supplier suggests) in 3% NGS overnight at 4 °C (300 µL per slide).
   2. Day 2
      1. 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 µL per slide).
      2. Wash slides 3x PBST, 2 min each. Treat slides with the DAB kit. Add 300 µL/slide for 3 min. Wash slides with dH₂O 2x (quick dips).
      3. 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).
      4. Dry slides on a paper towel under the hood for 10-20 min.
      5. Mount slides using 1 drop of mounting media, cover with coverslip. Label and store at RT.
      6. Image the slides under a microscope and quantify DAB signal with ImageJ⁷ (see section 4).
3. Immunostaining: Fluorescence
   1. On Day 1 follow steps described in step 2.2.1. Perform the following steps on Day 2.
   2. 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.
   3. Wash 3x in PBST, 2 min each. Mount slides using 1 drop of 4′,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 °C until fluorescence imaging.
4. Quantification using Image J
   1. Download the "Fiji" version of ImageJ, which includes the necessary plugins for DAB staining quantification.
   2. Open an image in Fiji and select Image → Color → Color Deconvolution.
   3. Select "H DAB" as the stain and then click OK. Three new images will appear. Select the image that contains only DAB staining.
   4. To quantify stromal staining only, use the ImageJ eraser function to remove the epithelium from the DAB image.
   5. Select Analyze → Measure (or Ctrl + M) and record the value.

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

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