This work has received funding from the Agence Nationale de Recherche (ANR), under a PRTS (Projet de Recherche Translationelle en Sant&#233;) grant No ANR-13-PRTS-0009 (V.B.) and from the European Union&#39;s Horizon 2020 research and innovation programme under the Marie Sk&#322;odowska-Curie grant agreement No 709104 (K.I.). The authors thank C&#233;line de Sousa for help with cell counting and histological processing.

All methods described here were carried out according to the tenets of the Declaration of Helsinki and followed international ethical requirements for human tissues. This was a prospective observational case control study. Informed consent was obtained from patients. No modifications to French standards of treatment or follow-up were made. Institutional Review Board (IRB) approval was obtained from the Patient Protection Committee, Ile-de-France V (14944).
1. Tissue Selection and Preparation
<ol>
	<li>Select donor corneas.
	<ol>
		<li>Transfer donor corneas stored in organ culture medium26 to dextran-supplemented organ culture medium for 3 days to allow deturgescence27 prior to FF-OCM imaging28 (see Table of Materials).</li>
	</ol>
	</li>
	<li>Prepare the samples.
	<ol>
		<li>Position the cornea, immersed in storage medium, in the sample holder with the epithelium facing up.</li>
		<li>Place a clean silica coverslip (provided with the sample holder) on top of the cornea and close the holder by gently turning its base until the sample is slightly flattened and immobilized under the coverslip providing a relatively even imaging surface. Take precautions to avoid any air bubbles.</li>
		<li>Apply a thick layer of ophthalmic or optical gel on the coverslip as the immersion medium.</li>
	</ol>
	</li>
</ol>
2. FF-OCM Initialization, Setup, and Image Acquisition
<ol>
	<li>Initialize the device.
	<ol>
		<li>Turn on the device by operating the power switch on the back of the device; illumination of a green LED on the front of the device indicates that the power is on.</li>
		<li>Turn on the dedicated computer and halogen light source by operating the power switch on the front.</li>
		<li>Launch the acquisition software (see Table of Materials) by double-clicking on the desktop shortcut.</li>
		<li>Ensure that the imaging stage is clear except for the movable tray. Then click &#34;OK&#34; to initialize the motors at the prompt.</li>
		<li>Pull out the tray and insert the sample holder in the dedicated container, then gently push back the tray.</li>
	</ol>
	</li>
	<li>Setup the device.
	<ol>
		<li>Enter a &#34;sample identifier&#34; in the designated and mandatory field; optionally enter a &#34;sample description&#34; and/or &#34;study description&#34;.</li>
		<li>Click &#34;Acquire macro image&#34; when ready, to create a snapshot of the sample that can be used for lateral positioning and navigation purposes later; once satisfied, validate the image at the prompt by clicking &#34;Yes&#34;, after which the device moves the sample tray underneath the objective and performs an auto-adjustment.</li>
		<li>Ensure that the microscope objective is well immersed in the optical gel before proceeding further.</li>
	</ol>
	</li>
	<li>Acquire the stacks.
	<ol>
		<li>Select the &#34;Explore&#34; tab to prepare an acquisition.
		<ol>
			<li>Before acquiring a stack of images, navigate to the center of the cornea, via translation of the joystick or manual selection on the screen (that is, by clicking and dragging the red square on the acquired macro image to the desired location).</li>
			<li>Vary the imaging depth via rotation of the joystick, adjustment of the slider, or manual keyboard input in the graphical user interface (GUI), and adjust the averaging value (an averaging of 40 is generally recommended for optimal corneal imaging). 
			NOTE: This is done to determine the thickness of the corneal sample and the averaging necessary to image through the entire corneal thickness while noting the first and last image location in depth. The bottom surface of the coverslip creates parallel interference fringes that are seen on the tomographic image (on the right-hand side of the GUI), which facilitate locating the corneal surface.</li>
			<li>Enter the corneal surface value or first image location in depth into the &#34;Depth&#34; field.</li>
		</ol>
		</li>
		<li>Select the &#34;Acquire tab&#34;, to acquire images.
		<ol>
			<li>Select the &#34;Slice spacing&#34; (the default and recommended setting is 1 &#181;m, matching the axial resolution of the device) and enter the corneal thickness value accordingly, under &#34;Number of slices&#34;.</li>
			<li>Review parameters and acquisition time, and when satisfied press &#34;OK&#34; to launch the acquisition.</li>
			<li>During the acquisition, avoid any contact with the table on which the FF-OCM is positioned.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Management of Acquired Images
<ol>
	<li>View and export images.
	<ol>
		<li>Launch the FF-OCM viewing software (see Table of Materials) by double-clicking on the desktop shortcut; acquired images (identified by the sample ID) appear in the &#34;Local Studies&#34; list.</li>
		<li>Select the study with corresponding sample ID, which contains both the macro image and acquired image stack, and &#34;export&#34; the latter by right-clicking on the series of images and selecting the &#34;DICOM&#34; format to retain the raw pixel data and metadata for further analysis.</li>
		<li>Display the 3D image stacks in en face and cross-sectional views using the multiplanar reconstruction (MPR) mode, by double-clicking on the series of images; navigate through the images (e.g., via mouse-wheel scroll or slider adjustment) and select representative en face and cross-sectional views of each stack.</li>
		<li>Export the selected views by clicking on the icon at the bottom right of the window, using the &#34;DICOM&#34; format.</li>
	</ol>
	</li>
	<li>Import images.
	<ol>
		<li>Open the image processing software (see Table of Materials) by double-clicking on the desktop shortcut, and navigate to the &#34;Bio-Formats&#34; &#34;Importer&#34; under &#34;Plugins&#34; to import DICOM images; make sure that &#34;Group files with similar names&#34; is selected in the &#34;Bio-Formats Import Options&#34; window.</li>
	</ol>
	</li>
</ol>
4. Image Analysis: Qualitatively and Quantitatively Assessment of Stromal Morphology and Features
<ol>
	<li>Assess stromal and Bowman&#39;s layer thickness.
	<ol>
		<li>Measure distances manually on corneal cross-sections, for example at five equally spaced points across the cross-section25.
		<ol>
			<li>Draw a line between two points of known distance (such as, from left to right of the entire image, that is according to the default field-of-view, 1,024 pixels or 780 &#181;m), go to &#34;Analyze&#34; and select &#34;Set Scale&#34;, and enter the &#34;Known distance&#34; and &#34;Unit of length&#34; in the appropriate fields and click &#34;OK&#34;.</li>
			<li>Draw a line between two points of unknown distance; read the length or distance measured directly from the status bar.</li>
		</ol>
		</li>
		<li>Record the mean and coefficient of variation (COV). Bowman&#39;s layer thickness less than 6.5 &#181;m and a COV greater than 18.6% have been associated with abnormal corneal stroma25.</li>
	</ol>
	</li>
	<li>Determine the keratocyte density.
	<ol>
		<li>Following convention of confocal microscopy: sum stromal en face images in groups of 7 to produce slices of comparable thickness. For this, go to the &#34;Image&#34; tab and select &#34;Reslice Z&#34; under &#34;Stacks&#34;.</li>
		<li>Take into account the differing (decreasing) cell density on progression through the stroma24,25. For this, the stroma may be considered as being composed of 4 regions according to depth: (1) the very anterior stroma below Bowman&#39;s layer, that is 2% of entire stromal thickness; the remaining stroma (that is, 98% of entire stromal thickness), with three zones of identical thickness: (2) anterior stroma, (3) mid stroma, and (4) posterior stroma.</li>
		<li>For further analysis, include all available en face slices for the very anterior stroma, 15 images for the anterior stroma, 5 images for the mid stroma, as well as 5 images for the posterior stroma (where the number of images per region required for a reliable count was determined by stepwise analysis29).</li>
		<li>On each en face image, select a 300 &#181;m x 300 &#181;m region of interest. To enhance nucleus visualization, invert the image, using &#34;Invert&#34; under the &#34;Edit&#34; tab, and adjust contrast and brightness. For the latter, go to &#34;Image&#34; and navigate to &#34;Brightness/Contrast&#34; under &#34;Analyze&#34;.</li>
		<li>Manually count cell nuclei, using for example the &#34;cell counter&#34; function25. For this, go to &#34;Plugins&#34; and navigate to &#34;Cell Counter&#34; under &#34;Analyze&#34;. Press &#8220;initialize&#8221; and then select a counter type.&#160;Then start counting the cell nuclei by clicking on dark oval features in the inverted image, considering those landing on an image border only for two of the four sides of the image25.</li>
		<li>Record the cell density in terms of area density, that is in cells/mm&#178; (divide the number of cells counted by 0.09, to convert from cells/90,000 &#181;m&#178; to cells/mm&#178;), following confocal microscopy convention where keratocyte densities have been shown to be lower in patients (e.g., with keratoconus) than in healthy subjects and to be correlated with disease severity25. 
		NOTE: Further clinical studies are necessary to determine whether a minimal threshold of keratocyte density is required to obtain a perfectly clear corneal graft after transplantation.</li>
	</ol>
	</li>
	<li>Assess the stromal reflectivity.
	<ol>
		<li>Generate mean intensity depth profiles of stromal image stacks, using for example the &#34;Z-axis profile&#34; function and/or custom software25,30,31. 
		NOTE:&#160;These are in fact amplitude depth profiles as FF-OCM measures the amplitude of light backscattered by the sample rather than its intensity. &#160;
		<ol>
			<li>Calculate the mean grey level for each en face stack.</li>
			<li>Subtract the minimum grey value and normalize by the maximum grey value.</li>
			<li>Display in log scale as a function of stromal depth (% of the stromal thickness).</li>
			<li>Approximate the resulting logarithmic profile by a linear regression line, which minimizes the sum of the squared residuals (least-squares fit).</li>
			<li>Record the R-square value (R&#178;) as a measure of linearity. Values below 0.94 could be an indication of corneal pathology25. 
			NOTE:&#160;To differentiate the inadequacy of a linear logarithmic regression model (or a mono-exponential decay model) due to presence of a pathology from mere measurement noise, signal analysis by Bayesian inference may be performed31. Also, in corneas with linear logarithmic (or mono-exponential) stromal depth profiles (e.g., represented by R-square values30&#160;or Birge ratios31 close to 1), the calculation of the photon mean-free path from the rate of signal decay may be used to further quantify the degree of transparency31.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Assess the visibility of other stromal features and disease indicators.
	<ol>
		<li>Check for the presence of scars, fibrotic tissue, lakes, or Vogt striae.</li>
		<li>Assess mean thickness of nerves in the stroma, if sufficiently visible for measurement24.
		<ol>
			<li>Select one en face image where the stromal nerve is most visible (generally in the mid-stromal region).</li>
			<li>Measure the thickness of the nerve, for example at five points24, then calculate the mean and standard deviation. Nerve thicknesses above 9 &#181;m may be an additional indicator for pathology, such as keratoconus24.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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Frontiers in Optics.</a> OSA Technical Digest (online), paper FW6A.6. , (2015).</li><li>Bocheux, R., Pernot, P., Borderie, V., Plamann, K., Irsch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measures+of+corneal+transparency,+derived+from+objective+analysis+of+depth-resolved+corneal+images,+demonstrated+with+full-field+optical+coherence+tomographic+microscopy.">Quantitative measures of corneal transparency, derived from objective analysis of depth-resolved corneal images, demonstrated with full-field optical coherence tomographic microscopy.</a> PLoS ONE. e0221707. 14, (2019).</li><li>Karimi, A. 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B., Kara, N., Yildirim, A., Bozkurt, E., Uslu, H., Demirok, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+corneal+microstructure+in+keratoconus:+a+confocal+microscopy+study.">Evaluation of corneal microstructure in keratoconus: a confocal microscopy study.</a> Am J Ophthalmol. 156, 885-893 (2013).</li><li>Erie, J. C., Patel, S. V., McLaren, J. W., Nau, C. B., Hodge, D. O., Bourne, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratocyte+density+in+keratoconus.+A+confocal+microscopy+study.">Keratocyte density in keratoconus. A confocal microscopy study.</a> Am J Ophthalmol. 134, 689-695 (2002).</li><li>Imre, L., Resch, M., Nagymihaly, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Konfokale+In-vivo-Hornhautmikroskopie+nach+Keratoplastik.">Konfokale In-vivo-Hornhautmikroskopie nach Keratoplastik.</a> Ophthalmology. 102, 140-146 (2005).</li><li>Niederer, R. L., Perumal, D., Sherwin, T., McGhee, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+scanning+in+vivo+confocal+microscopy+reveals+reduced+innervation+and+reduction+in+cell+density+in+all+layers+of+the+keratoconic+cornea.">Laser scanning in vivo confocal microscopy reveals reduced innervation and reduction in cell density in all layers of the keratoconic cornea.</a> Invest Ophthalmol Vis Sci. 49, 2964-2970 (2008).</li><li>Patel, S., McLaren, J., Hodge, D., Bourne, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+human+keratocyte+density+and+corneal+thickness+measurement+by+using+confocal+microscopy+in+vivo.">Normal human keratocyte density and corneal thickness measurement by using confocal microscopy in vivo.</a> Invest Ophthalmol Vis Sci. 42, 333-339 (2001).</li><li>Komuro, A., Hodge, D. O., Gores, G. J., Bourne, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death+during+corneal+storage+at+4+degrees.">Cell death during corneal storage at 4 degrees.</a> C. Invest Ophthalmol Vis Sci. 40, 2827-2832 (1999).</li><li>Gambato, C., Longhin, E., Catania, A. G., Lazzarini, D., Parrozzani, R., Midena, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+and+corneal+layers:+an+in+vivo+corneal+confocal+microscopy+study.">Aging and corneal layers: an in vivo corneal confocal microscopy study.</a> Graefes Arch Clin Exp Ophthalmol. 253, 267-275 (2015).</li></ol>

The authors have nothing to disclose.

The protocol described here for the qualitative and quantitative assessment of corneal donor stroma using FF-OCM is based on the histology-like analysis of macroscopic and microscopic features indicative of stromal condition, beyond the capabilities of spectral-domain OCT and confocal microscopy21,24,25, and enables differentiation of diseased from normal human tissues.
Aside from an excellent endothelial quality assessment of human donor corneas by means of specular microscopy, assessment of stromal quality is challenging in eye banks, and generally limited to a gross observation with slit-lamp biomicroscopy and/or light microscopy in current protocols. Lack of fine resolution with existing methods not only means that corneas with some stromal disease may be selected that compromise the result of keratoplasty, but also that corneas may be rejected for stromal opacities that are in fact constraint to anterior stroma or epithelial regions and could still be used for endothelial keratoplasty procedures14.
The current eye-bank protocol could be supplemented by the addition of FF-OCM, which due to its superior resolution, constitutes a powerful and non-invasive tool to complete the quality assessment of the cornea, especially the stroma (including Bowman&#39;s layer). Unlike during slit-lamp examination, the graft remains immersed in a closed chamber filled with storage medium throughout FF-OCM image acquisition, decreasing any potential risk of contamination.
For successful image acquisition with FF-OCM (see Table of Materials), it is important for the microscope objective to be well immersed in the optical gel that is applied on top of the coverslip of the sample holder (step 2.2.3). It is further recommended to regularly check the calibration of the device, a procedure also to be performed after unsuccessful auto-adjustment (step 2.2.2) and accessed via &#34;Tools and options&#34; in the acquisition software (see Table of Materials). The procedure, which involves the utilization of a calibration mirror in the sample holder, is the same as the usual sample preparation (see step 1.2) except that the optical gel should be applied on the mirror before positioning of the coverslip.
A series of donor corneal grafts, considered to have normal stroma as per existing eye-bank procedures, were used to describe the protocol in this manuscript and specifically demonstrate the suitability of FF-OCM for precise and reliable assessment of donor stromal quality. These normal donor corneas were compared with pathological corneas immersed in storage medium, showing that the histology-like analysis made possible with FF-OCM of several stromal features (illustrated in Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, and Figure 8) in corneal grafts allows distinguishing diseased from normal human corneal tissues.
Aside from morphological changes, such as the presence of scars (Figure 5 and Figure 7), fibrotic tissue (Figure 8), lakes (Figure 2), Vogt striae (Figure 4), or increased stromal nerve diameter (Figure 4), typical stromal features are present in diseased corneas. Stromal parameters particularly relevant in the stromal quality assessment appear to be Bowman&#39;s layer thickness and its variability, and stromal reflectivity. Critical steps within the protocol are thus steps 4.1 and 4.3.
While being secreted during human corneal development, Bowman&#39;s layer, in particular, becomes distinct by 19 weeks of gestation and never repairs after birth32. Damage to Bowman&#39;s layer is thus irreversible and serves as an ideal indicator of previous stromal damage in donor corneal tissue, including damage caused by refractive surgery, infectious keratitis, keratoconus. Such corneal diseases, which constitute contraindications for donor corneal usage, are associated with decreased and variable Bowman&#39;s layer thickness due to interruption and scarring (Figure 5), and are likely to be missed by current eye-bank protocols when the donor history is not precisely known.
Although corneal transparency is impaired after donor death due to post mortem corneal edema, the amount of backscattered light, or stromal reflectivity is expected to decrease exponentially with depth in the stroma (see Figure 3 and Figure 4A); as a result, the logarithm of normalized stromal reflectivity will be a linear function of stromal depth in normal donor corneas, represented by R-square values close to 1. Conversely, the presence of macroscopic features is associated with non-linear logarithmic depth profiles and indicative of stromal disease (Figure 4B and Figure 7)25.
Since keratocyte density is responsible for stromal collagen fibril and extracellular matrix synthesis and renewal, it appears reasonable to assume that keratocyte density is another relevant parameter for assessing donor stromal quality, and that tissues exhibiting very low keratocyte counts should not be transplanted. The protocol therefore includes a precise and reliable method to measure keratocyte density from FF-OCM images that can be easily used in eye banks25 and follows the convention of confocal microscopy. Note that with FF-OCM, keratocyte density may also be determined by counting keratocytes directly in the cross-sectional view33, a potential advantage over confocal microscopy, which requires keratocytes to be counted on multiple en face slices. However, unlike in living patients, where keratocyte densities have been demonstrated to be lower in disease patients than in normal controls34,35,36,37 and to correlate with disease severity34,38, this was not the case in human ex vivo tissue samples25, and further studies are necessary to determine whether a minimal number of keratocytes is required in donor corneas to result in good visual recovery after transplantation. Low keratocyte density in donor tissue as in pathological tissue could be explained by aging, post mortem loss of cells induced by ischemia, and/or storage of donor tissue27,39,40,41. It should also be pointed out that the normal donor corneas that were obtained and imaged in this protocol were either stored and edematous or de-swelled, or had been discarded by the eye bank before transplantation because of poor endothelial quality according to the standards of the EU Eye Bank Association. Were FF-OCM imaging along with the described protocol to be included in the eye bank setting, the corneas would typically be assessed in a fresher state than was possible here, which may affect the keratocyte densities.
The protocol described here for the stromal quality analysis could be extended for assessment of Descemet&#39;s membrane, which can also be resolved with FF-OCM in terms of thickness and structure21,24. This may prove useful for selection of tissues for Descemet&#39;s membrane endothelial keratoplasty, where thin Descemet&#39;s membranes may be more difficult to separate from the stroma.
In conclusion, FF-OCM enables precise and reliable assessment of human donor corneal stroma during storage. By improving the graft quality, the addition of this protocol to current eye-banking procedures has the potential to improve the screening and selecting of donor tissues, and hence the results of keratoplasty. Real-life integration of the FF-OCM device into the eye-bank routine should be facilitated by recent technological updates, including faster image acquisition and larger field of view thanks to the development of a custom CMOS camera, and the design of custom sterile disposable cassettes for cornea storage and handling during imaging.

Corneal diseases are amongst the major causes of blindness worldwide1. Some diseases can only be treated surgically, often involving the replacement of part (i.e., lamellar keratoplasty) or the entire (i.e., penetrating keratoplasty) diseased cornea, by donated tissue, the graft, taken from a recently deceased individual. For corneal diseases that do not affect the endothelium (e.g., keratoconus, stromal scars after infectious keratitis, trauma, and stromal dystrophies), deep anterior lamellar keratoplasty (DALK) is currently considered as the surgical technique of choice2,3,4,5. This technique makes possible the preservation of the recipient&#39;s corneal endothelium, by replacing only the central corneal epithelium and stroma, which is associated with a lower incidence of graft rejection, absence of endothelial rejection, lower endothelial cell loss, and favorable cost-effectiveness ratio6,7,8,9,10,11. DALK further allows corneas with less than optimal endothelium quality to be used as grafts, as this compromised layer will not be transplanted12. Conversely, quality of donor corneal stroma is likely to be the major limiting factor for graft success and vision recovery because the stroma is the only donor corneal layer to remain, while the donor epithelium will be replaced by recipient epithelium. Unfortunately, means to assess donor corneal stroma in eye banks are limited. They usually include slit-lamp examination of the donor eyeball when tissue retrieval is made by enucleation and light microscope examination of the donor stroma13. Some eye banks have started supplementing such standard procedures using Fourier-domain optical coherence tomography (FD-OCT)14.
Ophthalmic optical coherence tomography (OCT), an optical analogue to ultrasound imaging15, uses interference of broadband or tunable light to generate optical sections of the retina16 and anterior segment17. In time-domain OCT, the basis of early clinical systems, the position of a reference mirror is altered, so that interference patterns show up whenever the reference beam has traveled almost the same amount of time as the beam reflected at the various tissue interfaces, with A-scans being generated as a function of time. In FD-OCT (also called spectral- or frequency-domain OCT), the basis of most modern clinical systems, the reference mirror is fixed at one position and an individual A-scan, with all interference patterns mixed together, is acquired at a time, and dissected apart via Fourier analysis.
While clinical (time or spectral-domain) OCT systems permit cross-sectional views of the cornea and the detection of stromal opacities at a higher axial resolution than slit-lamp biomicroscopy, their lateral resolution is limited. Confocal microscopy18 allows examination of the cornea at a lateral resolution approaching histological detail, but is limited axially.
Full-field optical coherence tomographic microscopy (FF-OCT or FF-OCM)19,20 combines elements of both confocal microscopy and OCT, achieving a lateral resolution comparable to the axial resolution of about 1 &#181;m. More specifically, FF-OCM uses incoherent broadband light sources (e.g., a halogen lamp) and high numerical aperture optics to acquire en face 2D tomographic images without lateral scanning. By scanning in the depth direction, FF-OCM enables non-invasive 3D imaging of fresh or fixed ex vivo biological tissue samples. It has been used to image the cornea21,22,23. By providing both high-resolution en face and cross-sectional views, FF-OCM provides information on both the histological structure and cellular details of the cornea. In fact, FF-OCM has been shown to provide structural information superior to histology and was able to identify more disease indicators as was possible with the combination of spectral-domain OCT and confocal microscopy24,25.
Here we describe a protocol for the qualitative and quantitative assessment of corneal donor stroma using FF-OCM. The method is based on the histology-like analysis of macroscopic and microscopic features indicative of stromal condition, including three quantitative stromal parameters (i.e., Bowman&#39;s layer thickness and its variability, and stromal reflectivity). The described protocol is therefore applied to normal and abnormal cornea tissues and permits differentiation of diseased from normal human corneal tissues.

<table><tbody><tr><td>Light-CT Scanner</td><td>LLTech, France</td><td>http://www.lltechimaging.com/products-applications/products/</td><td>FF-OCM device used in this manuscript for imaging</td></tr><tr><td>CorneaJet</td><td>EuroBio, France</td><td>http://www.eurobio-cornea.com/en/corneamax-10-100-ml-xml-352-822.html</td><td>Organ culture medium in which donor corneas are stored</td></tr><tr><td>CorneaMax</td><td>EuroBio, France</td><td>http://www.eurobio-cornea.com/en/corneajet-10-50-ml-xml-352-823.html</td><td>Dextran-supplemented organ culture medium used for deturgescence&amp;nbsp;</td></tr><tr><td>Fiji (ImageJ)</td><td>National Institute of Health, Bethesda, MD, USA</td><td>https://fiji.sc/</td><td>Open source image processing software</td></tr><tr><td>Matlab</td><td>Mathworks, Inc., Natick, MA, USA</td><td>https://www.mathworks.com/products/matlab.html</td><td>Mathematical computing software</td></tr></tbody></table>

full-field optical coherence microscopy for histology-like analysis of stromal features in corneal grafts

The quality of donor corneal stroma, which makes up about 90% of total corneal thickness, is likely to be one of the main, if not the major, limiting factor(s) for success of deep anterior lamellar and penetrating keratoplasty. These are surgical procedures that involve replacing part or all of the diseased corneal layers, respectively, by donated tissue, the graft, taken from a recently deceased individual. However, means to evaluate stromal quality of corneal grafts in eye banks are limited and lack the capability of high-resolution quantitative assessment of disease indicators. Full-field optical coherence microscopy (FF-OCM), permitting high-resolution 3D imaging of fresh or fixed ex vivo biological tissue samples, is a non-invasive technique well suited for donor cornea assessment. Here we describe a method for the qualitative and quantitative analysis of corneal stroma using FF-OCM. The protocol has been successfully applied to normal donor corneas and pathological corneal buttons, and can be used to identify healthy and pathologic features on both the macroscopic and microscopic level, thereby facilitating the detection of stromal disorders that could compromise the outcome of keratoplasty. By improving the graft quality control, this protocol has the potential to result in better selection (and rejection) of donor tissues and hence decreased graft failure.

The FF-OCM device (see Table of Materials)28 and general setup used in this manuscript is shown in Figure 1. Figure 2 shows a swelled human donor cornea after storage in organ culture medium, giving a pathophysiological model of edematous cornea and preventing FF-OCM image acquisition through the entire corneal thickness due to limited penetration depth. Transfer to dextran-supplemented organ culture medium causes deswelling and results in donor corneas of normal thickness, as represented in Figure 3. Diseased corneas can be recognized by morphological changes and typical stromal features, including a decreased and variable thickness of the stroma (Figure 4) and/or Bowman&#39;s layer (Figure 5). Assessment of the keratocyte density (Figure 6) and stromal reflectivity (Figure 7) may further aid in the histology-like analysis and differentiation of normal from pathologic corneal tissues with FF-OCM, beyond the capabilities of clinical OCT systems (Figure 8).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57104/57104fig1.jpg" /> 
Figure 1: Schematic and photographs of the general setup and FF-OCM device used in this work. (A) Photograph of the FF-OCM device (see Table of Materials)28. (B) Schematic and optical setup of the device. (C) Photograph of a human donor cornea in the sample holder. (D) Zoom on the immersion objective and the sample holder. <a href="https://cloudflare.jove.com/files/ftp_upload/57104/57104fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57104/57104fig2.jpg" /> 
Figure 2: Organ-cultured normal human cornea before deturgescence. Cross-sectional (A) and en face view (B, slice through the stroma) of swelled (&#34;edematous&#34;) cornea caused by storage in organ culture medium, where lakes can be seen as dark areas (as indicated by arrows). Corneal thickness has doubled to over 1,100 &#181;m, preventing image acquisition through the entire depth. <a href="https://cloudflare.jove.com/files/ftp_upload/57104/57104fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57104/57104fig3.jpg" /> 
Figure 3: Organ-cultured normal human cornea after deturgescence. Cross-sectional view through the entire cornea (A) and en face stromal view (B) of de-swelled cornea immersed in dextran-supplemented medium, showing regularly distributed hyper-reflective (white) keratocytes. <a href="https://cloudflare.jove.com/files/ftp_upload/57104/57104fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57104/57104fig4.jpg" /> 
Figure 4: Overall assessment of stromal morphology and features, including stromal thickness. Compared with normal human cornea (A), corneas with keratoconus (B) feature decreased and variable stromal thickness, along with numerous, parallel Vogt striae that are observable as dark vertical bands in cross-sectional views, and thick stromal nerves that may be found in mid-stromal en face slices (C). <a href="https://cloudflare.jove.com/files/ftp_upload/57104/57104fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57104/57104fig5.jpg" /> 
Figure 5: Assessment of Bowman&#39;s layer thickness, which is delineated anteriorly by the hyper-reflective epithelial basement membrane and posteriorly by the hyper-reflective keratocytes in the very anterior stroma. Compared with a normal human cornea (A), pathologic cornea (e.g., keratoconus) (B) features decreased and variable Bowman&#39;s layer thickness due to interruption and scarring. <a href="https://cloudflare.jove.com/files/ftp_upload/57104/57104fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57104/57104fig6.jpg" /> 
Figure 6: Assessment of keratocyte density. (A) Keratocyte nuclei are manually counted on enhanced and inverted en face images after selection of a 300 &#181;m x 300 &#181;m region of interest. (B) Counted nuclei are indicated by arrows. <a href="https://cloudflare.jove.com/files/ftp_upload/57104/57104fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57104/57104fig7.jpg" /> 
Figure 7: Assessment of stromal reflectivity. The amount of backscattered light decreases (exponentially) with depth in the stroma of normal donor corneas (see Figure 3 and Figure 4A), resulting in linear logarithmic depth profiles, represented by R-square values close to 1 (green trace). This may not hold true for pathological corneas featuring stromal regions of increased light backscatter (see Figure 4B). The presence of such macroscopic features, as in the example of a cornea with stromal scar after infectious keratitis (depicted in the inset), will thus create non-linear logarithmic intensity depth profiles, represented by an R-square value below a certain threshold (e.g., lower than 0.9425; red trace). Note that logarithmic amplitude depth profiles are in fact shown as FF-OCM measures the amplitude of light backscattered by the sample rather than its intensity. &#160;<a href="https://cloudflare.jove.com/files/ftp_upload/57104/57104fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/57104/57104fig8.jpg" /> 
Figure 8: Demonstration of benefits of FF-OCM over histology and spectral-domain OCT. (A) Histology and (B) corresponding spectral-domain-OCT image acquired on the same cornea in vivo prior to keratoplasty. (C) Corresponding FF-OCM cross section of the ex vivo corneal button post-keratoplasty, illustrating superior resolution compared to the clinical spectral-domain OCT images. The fibrosis is also more clearly visible in FF-OCM images than in the histology. The high density of keratocytes in the upper stroma can clearly be seen in both the (C) cross-sectional and (D) en face views. <a href="https://cloudflare.jove.com/files/ftp_upload/57104/57104fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Full-Field Optical Coherence Microscopy for Histology-Like Analysis of Stromal Features in Corneal Grafts at JoVE.com

Full-Field Optical Coherence Microscopy for Histology-Like Analysis of Stromal Features in Corneal Grafts

We describe use of full-field optical coherence microscopy as a method for high quality assessment of corneal donor stroma. This protocol can be used to identify features indicative of health or disease, and is aimed at improving the screening and selection of donor tissues, and hence the results of keratoplasty.

The quality of donor corneal stroma, which makes up about 90% of total corneal thickness, is likely to be one of the main, if not the major, limiting ...

full-field-optical-coherence-microscopy-for-histology-like-analysis

Research

JoVE Journal

Immunology and Infection

1.6K Views.  Sorbonne University, UM 80 / INSERM, UMR_S 968 / CNRS, UMR_7210. Deze methode kan de screening en selectie van hoornvliesdonorweefsels in oogbanken verbeteren, de resultaten van hoornvliestransplantatie verbeteren. Het belangrijkste voordeel van deze techniek is de hoge resolutie beoordelingsvermogen en dat het hoornvlies tijdens de beeldacquisitie ondergedompeld blijft in een gesloten kamer gevuld met opslagmedium, waardoor het potentiële risico op besmetting wordt verminderd. Maelle Vilbert, promovendus van ons laboratorium, zal de procedure demonstrere...

Video: Full-Field Optical Coherence Microscopy for Histology-Like Analysis of Stromal Features in Corneal Grafts

Corneal Confocal Microscopy: A Novel Non-invasive Technique to Quantify Small Fibre Pathology in Peripheral Neuropathies

The accurate quantification of peripheral neuropathy is important to define at risk patients, anticipate deterioration, and assess new therapies. Conventional methods assess neurological deficits and electrophysiology and quantitative sensory testing quantifies functional alterations to detect neuropathy. However, the earliest damage appears to be to the small fibres and yet these tests primarily assess large fibre dysfunction and have a limited ability to demonstrate regeneration and repair. The only techniques which allow a direct examination of unmyelinated nerve fibre damage and repair are sural nerve biopsy with electron microscopy and skin-punch biopsy. However, both are invasive procedures and require lengthy laboratory procedures and considerable expertise. Corneal Confocal microscopy is a non-invasive clinical technique which provides in-vivo imaging of corneal nerve fibres. We have demonstrated early nerve damage, which precedes loss of intraepidermal nerve fibres in skin biopsies together with stratification of neuropathic severity and repair following pancreas transplantation in diabetic patients. We have also demonstrated nerve damage in idiopathic small fibre neuropathy and Fabry's disease.

Corneal Confocal microscopy is a non-invasive clinical technique which may be used to quantify C fibre damage to diagnose and stratify patients with increasing neuropathic severity.

The accurate quantification of peripheral neuropathy is important to define at risk patients, anticipate deterioration, and assess new therapies. ...

Medicine

Corneal Donor Tissue Preparation for Endothelial Keratoplasty

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full thickness corneal transplantation has been the treatment to restore sight in those limited by corneal disease. Some disadvantages to this approach include a high degree of post-operative astigmatism, lack of predictable refractive outcome, and disturbance to the ocular surface. The development of Descemet's stripping endothelial keratoplasty (DSEK), transplanting only the posterior corneal stroma, Descemet's membrane, and endothelium, has dramatically changed treatment of corneal endothelial disease. DSEK is performed through a smaller incision; this technique avoids 'open sky' surgery with its risk of hemorrhage or expulsion, decreases the incidence of postoperative wound dehiscence, reduces unpredictable refractive outcomes, and may decrease the rate of transplant rejection3-6.
Initially, cornea donor posterior lamellar dissection for DSEK was performed manually1 resulting in variable graft thickness and damage to the delicate corneal endothelial tissue during tissue processing. Automated lamellar dissection (Descemet's stripping automated endothelial keratoplasty, DSAEK) was developed to address these issues. Automated dissection utilizes the same technology as LASIK corneal flap creation with a mechanical microkeratome blade that helps to create uniform and thin tissue grafts for DSAEK surgery with minimal corneal endothelial cell loss in tissue processing.
Eye banks have been providing full thickness corneas for surgical transplantation for many years. In 2006, eye banks began to develop methodologies for supplying precut corneal tissue for endothelial keratoplasty. With the input of corneal surgeons, eye banks have developed thorough protocols to safely and effectively prepare posterior lamellar tissue for DSAEK surgery. This can be performed preoperatively at the eye bank. Research shows no significant difference in terms of the quality of the tissue7 or patient outcomes8,9 using eye bank precut tissue versus surgeon-prepared tissue for DSAEK surgery. For most corneal surgeons, the availability of precut DSAEK corneal tissue saves time and money10, and reduces the stress of performing the donor corneal dissection in the operating room. In part because of the ability of the eye banks to provide high quality posterior lamellar corneal in a timely manner, DSAEK has become the standard of care for surgical management of corneal endothelial disease.
The procedure that we are describing is the preparation of the posterior lamellar cornea at the eye bank for transplantation in DSAEK surgery (Figure 1).

Endothelial corneal transplantation is a surgical technique for treatment of posterior corneal diseases. Mechanical microkeratome dissection to prepare tissue results in thinner, more symmetric grafts with less endothelial cell loss and improved outcomes. Dissections can be performed at the eye bank prior to corneal transplantation surgery.

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full ...

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, high-throughput imaging modalities utilizing bright field or fluorescence detection, are unable to resolve the overall 3D structure of the tumor spheroid due to limited light penetration, diffusion of fluorescent dyes and depth-resolvability. Recently, our lab demonstrated the use of optical coherence tomography (OCT), a label-free and non-destructive 3D imaging modality, to perform longitudinal characterization of multicellular tumor spheroids in a 96-well plate. OCT was capable of obtaining 3D morphological and physiological information of tumor spheroids growing up to about 600 &#181;m in height. In this article, we demonstrate a high-throughput OCT (HT-OCT) imaging system that scans the whole multi-well plate and obtains 3D OCT data of tumor spheroids automatically. We describe the details of the HT-OCT system and construction guidelines in the protocol. From the 3D OCT data, one can visualize the overall structure of the spheroid with 3D rendered and orthogonal slices, characterize the longitudinal growth curve of the tumor spheroid based on the morphological information of size and volume, and monitor the growth of the dead-cell regions in the tumor spheroid based on optical intrinsic attenuation contrast. We show that HT-OCT can be used as a high-throughput imaging modality for drug screening as well as characterizing biofabricated samples.

Optical coherence tomography (OCT), a three-dimensional imaging technology, was used to monitor and characterize the growth kinetics of multicellular tumor spheroids. Precise volumetric quantification of tumor spheroids using a voxel counting approach, and label-free dead tissue detection in the spheroids based on intrinsic optical attenuation contrast, were demonstrated.

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, ...

Cancer Research

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide suffering from corneal vision loss1. To address the demand for viable human corneas, significant progress in three-dimensional (3D) tissue engineering has been made2,3,4. These cornea models range from simple monolayer systems to multilayered models, leading to 3D full-thickness corneal equivalents2.
However, the use of a 3D tissue-engineered cornea in the context of in vitro disease models studied to date lacks resemblance to the multilayered 3D corneal tissue structure, function, and the networking of different cell types (i.e., nerve, epithelium, stroma, and endothelium)2,3. In addition, the demand for in vitro cornea tissue models has increased in an attempt to reduce animal testing for pharmaceutical products. Thus, more sophisticated models are required to better match systems to human physiological requirements, and the development of a model that is more relevant to the patient population is absolutely necessary. Given that multiple cell types in the cornea are affected by diseases and dystrophies, such as Keratoconus, Diabetic Keratopathy, and Fuchs, this model includes a 3D co-culture model of primary human corneal fibroblasts (HCFs) from healthy donors and neurons from the SH-SY5Y cell line. This allows us for the first time to investigate the interactions between the two cell types within the human corneal tissue. We believe that this model could potentially dissect the underlying mechanisms associated with the stromal-nerve interactions of corneal diseases that exhibit nerve damages. This 3D model mirrors the basic anatomical and physiological nature of the corneal tissue in vivo and can be used in the future as a tool for investigating corneal defects as well as screening the efficacy of various agents before animal testing.

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

This protocol describes a novel three-dimensional in vitro model, where corneal stromal cells and differentiated neuronal cells are cultured together to assist in the examination and understanding of the interactions of the two cell types.

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide ...

Developmental Biology

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as Aniridia or Steven Johnson Syndrome as well as by external factors such as chemical burns or radiation. Current treatments are (i) the use of corneal grafts and (ii) the use of stem cell expanded in the laboratory and delivered on carriers (e.g., amniotic membrane); these treatments are relatively successful but unfortunately they can fail after 3-5 years. There is a need to design and manufacture new corneal biomaterial devices able to mimic in detail the physiological environment where stem cells reside in the cornea. Limbal stem cells are located in the limbus (circular area between cornea and sclera) in specific niches known as the Palisades of Vogt. In this work we have developed a new platform technology which combines two cutting-edge manufacturing techniques (microstereolithography and electrospinning) for the fabrication of corneal membranes that mimic to a certain extent the limbus. Our membranes contain artificial micropockets which aim to provide cells with protection as the Palisades of Vogt do in the eye.

We report a technique for the fabrication of micropockets within electrospun membranes in which to study cell behavior. Specifically, we describe a combination of microstereolithography and electrospinning for the production of PLGA (Poly(lactide-co-glycolide)) corneal biomaterial devices equipped with microfeatures.

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as ...

Bioengineering

Investigating Scarless Tissue Regeneration in Embryonic Wounded Chick Corneas

Chick embryonic corneal wounds display a remarkable capacity to fully and rapidly regenerate, whereas adult wounded corneas experience a loss of transparency due to fibrotic scarring. The tissue integrity of injured embryonic corneas is intrinsically restored with no detectable scar formation. Given its accessibility and ease of manipulation, the chick embryo is an ideal model for studying scarless corneal wound repair. This protocol demonstrates the different steps involved in wounding the cornea of an embryonic chick in ovo. First, eggs are windowed at early embryonic ages to access the eye. Second, a series of in ovo physical manipulations to the extraembryonic membranes are conducted to ensure access to the eye is maintained through later stages of development, corresponding to when the three cellular layers of the cornea are formed. Third, linear cornea wounds that penetrate the outer epithelial layer and the anterior stroma are made using a microsurgical knife. The regeneration process or fully restored corneas can be analyzed for regenerative potential using various cellular and molecular techniques following the wounding procedure. Studies to date using this model have revealed that wounded embryonic corneas display activation of keratocyte differentiation, undergo coordinated remodeling of ECM proteins to their native three-dimensional macrostructure, and become adequately re-innervated by corneal sensory nerves. In the future, the potential impact of endogenous or exogenous factors on the regenerative process could be analyzed in healing corneas by using developmental biology techniques, such as tissue grafting, electroporation, retroviral infection, or bead implantation. The current strategy identifies the embryonic chick as a crucial experimental paradigm for elucidating the molecular and cellular factors coordinating scarless corneal wound healing.

The present protocol demonstrates the different steps involved in wounding the cornea of an embryonic chick in ovo. The regenerating or fully restored corneas can be analyzed for regenerative potential using various cellular and molecular techniques following the wounding procedure.&#160;

Chick embryonic corneal wounds display a remarkable capacity to fully and rapidly regenerate, whereas adult wounded corneas experience a loss of ...

Three Different Protocols of Corneal Collagen Crosslinking in Keratoconus: Conventional, Accelerated and Iontophoresis

Keratoconus is a bilateral and progressive corneal ectasia. In order to slow down its progression, corneal collagen cross-linking (CXL) has recently been introduced as an efficient treatment option. In biological and chemical sciences, crosslinking refers to new chemical bonds formed between reactive molecules. Hence, the aim of corneal collagen CXL is to synthetically increase the formation of crosslinks between collagen fibrils in the corneal stroma. Despite the fact that the efficiency of the conventional CXL (C-CXL) protocol has already been shown in several clinical studies, it might benefit from improvements in duration of the procedure and removal of corneal epithelium. Hence, in order to provide a coherent evaluation of two new and optimized CXL protocols, we studied keratoconus patients who had undergone one of the three CXL treatments: iontophoresis (I-CXL), accelerated CXL (A-CXL), and conventional CXL (C-CXL). A-CXL is a 6 time faster CXL procedure using a ten time higher UVA irradiance but still including an epithelium removal. Iontophoresis is a transepithelial non-invasive technique in which a small electric current is applied to improve riboflavin penetration throughout the cornea. Using anterior segment optical coherence tomography (AS OCT) and in vivo confocal microscopy (IVCM), we conclude that regarding the depth of treatment penetration, conventional CXL protocol remains the standard for treating progressive keratoconus. Accelerated CXL seems to be a quick, effective and safe alternative to treat thin corneas. The use of iontophoresis is still being investigated and should be considered with greater caution.

Corneal collagen cross-linking (CXL) is the only conservative treatment currently available to halt keratoconus progression by improving the biomechanical rigidity of the corneal stroma. The aim of this manuscript is to highlight the methods of three different protocols of CXL: conventional CXL (C-CXL), accelerated CXL (A-CXL), and iontophoresis CXL (I-CXL).

Keratoconus is a bilateral and progressive corneal ectasia. In order to slow down its progression, corneal collagen cross-linking (CXL) has recently been ...

A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with conjunctiva. Patients suffer from recurrent corneal defects, pain, inflammation, and loss of vision.
Previously, a murine model of LSCD was described and compared to two other models. The goal was to produce a consistent mouse model of LSCD that both mimics the phenotype in humans and lasts long enough to make it possible to study the disease pathophysiology and to evaluate new treatments. Here, the technique is described in more detail.
A motorized tool with a rotating burr has been designed to remove the rust rings from the corneal surface or to smooth the pterygium bed in patients. It is a suitable device to create the desired LSCD model. It is a readily available, easy-to-use tool with a fine tip that makes it appropriate for working on small eyes, as in mice. Its application prevents unnecessary trauma to the eye and it does not result in unwanted injuries, as often is the case with chemical injury models. As opposed to a blunt scraper, it removes the epithelium with the basement membrane. In this protocol, the limbal area was abraded two times, and then the whole corneal epithelium was shaved from limbus to limbus. To avoid stroma injury, care was taken not to brush the corneal surface once the epithelium was already removed.

The following article provides an easy, reproducible technique to effectively create a sustainable mouse model of limbal stem cell deficiency (LSCD). This animal model is useful in testing and comparing the efficacy of treatments for limbal stem cell diseases.

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

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.

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 ...

A Human Corneal Organ Culture Model of Descemet's Stripping Only with Accelerated Healing Stimulated by Engineered Fibroblast Growth Factor 1

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the whole cornea or Descemet's membrane. Recent developments in ocular surgery have established Descemet's Stripping Only (DSO), a surgical technique in which a central circle of guttae-dense Descemet's membrane is removed to allow for the migration of CECs onto the smooth stroma, restoring function and vision to the cornea. While this potential treatment option is of high interest in the field of ophthalmic research, no successful ex vivo models of DSO have been established and clinical data is limited. This work presents a novel wound-healing model simulating DSO in human donor corneas. Using this approach to evaluate the efficacy of the human engineered FGF1 (NM141), we found that treatment accelerated healing via stimulation of migration and proliferation of CECs. This finding was confirmed in 11 pairs of human corneas with signs of dystrophy reported by the eye banks in order to verify that these results can be replicated in patients with Fuchs' Dystrophy, as the target population of the DSO procedure.

Descemet's Stripping Only is an experimental procedure wherein patients with central corneal guttae resulting from Fuchs Endothelial Corneal Dystrophy have Descemet's membrane stripped for peripheral cells to regenerate the endothelial layer. We present novel methodology simulating DSO in dystrophic human corneas ex vivo with accelerated healing stimulated by eFGF1 (NM141).

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the ...

Full-Field Optical Coherence Microscopy for Histology-Like Analysis of Stromal Features in Corneal Grafts

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