Thanks to No&#233;mie Gallorini for her assistance during the preparation of Figure 7. This work was supported by a project grant awarded to DH by the National Health and Medical Research Council of Australia (Project Grant 1099922), and by supplementary funding received from the Queensland Eye Institute Foundation.

Human corneas with donor consent for research were obtained from the Queensland Eye Bank and used with ethics approval from the Metro South Hospital and Health Service&#39;s Human Research Ethics Committee (HREC/07/QPAH/048). Sheep corneas were obtained from euthanized animals at the Herston Medical Research Facility of the University of Queensland under a tissue sharing agreement.
1. Preparation of dissection tools
<ol>
	<li>Sterilize two pairs of number 4 watchmaker forceps by either soaking them in a solution of 70% ethanol for 5 min or by autoclaving them.</li>
</ol>
2. Preparation of culture medium and tissue culture plates
<ol>
	<li>Prepare 100 mL of culture medium containing Opti-MEM 1 (1x) + GlutaMAX-1, 5% fetal bovine serum and 100 U/mL Pen/Strep. This culture medium is adequate for sheep corneal endothelial cell cultures, however, for human corneal endothelial cell cultures the medium should be supplemented with 50 &#181;g/mL bovine pituitary extract, 0.08% chondroitin sulphate, 200 &#181;g/mL calcium chloride and 0.3 mM L-ascorbic acid 2-phosphate. Culture medium can be stored in the dark at 4 &#176;C for two weeks.</li>
	<li>Coat the wells of a 6-well tissue culture plate with Attachment Factor using the manufacturer&#39;s instructions and then add 1 mL of culture medium to each coated well. Prepare one well for each cornea.</li>
</ol>
3. Explant dissection and cell culture procedure
<ol>
	<li>Place the cornea, endothelium side up, into a sterile Petri dish on the stage of a dissecting microscope. Adjust the illumination so that the corneal surface is well-lit with sufficient contrast to highlight the endothelial layer. Adjust the zoom so that the edge and some central corneal endothelium is in view.</li>
	<li>Use sterilized watchmaker forceps to gently lift and tear Descemet&#39;s membrane away from the underlying stroma (Figure 1). The membrane will detach from the stroma as a strip that immediately curls up. Place the strip into one well of a 6-well tissue culture plate that has been coated with Attachment Factor and contains 1 mL of culture medium. The lid of the tissue culture plate should be kept on at all times, except when explants are being added to it, to reduce the risk of contamination.</li>
	<li>Remove strips of Descemet&#39;s membrane from the extreme periphery of the endothelium first and then from central regions later. Place all strips from one cornea into a single well within the 6-well plate.</li>
	<li>Incubate the explants in a humidified cell culture incubator set at 5% CO2 and 37 &#176;C. Leave the culture undisturbed for 2 days to allow the explants to settle and attach to the plate surface. Typically, up to one third of explants fail to attach to the plate.</li>
	<li>Remove the medium and any unattached explants from the culture after 4 days and gently add 2 mL of fresh culture medium taking care not to disturb the attached explants. Culture medium should be changed twice per week.</li>
</ol>
4. Continuous production of corneal endothelial cells by serial explant culture
NOTE: Explants can be transferred to fresh tissue culture plates after 10 days to establish additional corneal endothelial cell cultures.
<ol>
	<li>Place the explant culture onto the stage of a dissecting microscope and adjust the illumination and zoom so that the explants are visible.</li>
	<li>Using sterilized watchmaker forceps, gently pluck each explant from its culture plate and transfer it to a fresh well of a 6-well tissue culture plate that has been coated with Attachment Factor and contains 1 mL of culture medium.</li>
	<li>Allow the explants to settle onto the surface of their new plate for 4 days before replacing the culture medium with 2 mL of fresh culture medium. Change culture medium changed twice per week.</li>
</ol>
5. Growing corneal endothelial cells on glass coverslips for immunofluorescence analyses
NOTE: Cell cultures that are destined to be analyzed using immunofluorescence should be established on glass coverslips that can be mounted onto glass microscope slides following the staining procedure.
<ol>
	<li>Sterilize a pack of round glass coverslips of 13 mm diameter in an autoclave or by soaking in 70% ethanol for 15 min followed by three rinses in phosphate-buffered saline (PBS).</li>
	<li>Place individual coverslips into the wells of a 24-well tissue culture plate, coat them with Attachment Factor, and then add 0.5 mL of culture medium to each well.</li>
	<li>Using sterilized watchmaker forceps remove explants from their tissue culture plates and transfer them to wells containing coverslips. Allow the explants to settle onto the coverslips for 4 days before changing the medium.</li>
	<li>After the required incubation period, fix and stain the coverslip cultures within their culture wells. Mount the stained coverslips onto glass microscope slides for analysis under a fluorescence microscope.</li>
</ol>
6. Subculture of corneal endothelial cells using Dispase II
NOTE: Large fibroblastic cells can be selectively removed from explant cultures in 6-well plates before subculturing using this procedure. If all cells are to be subcultured, do not perform steps 6.2 to 6.4. The aim of this procedure is to transfer the cells to fresh plates while maintaining their cell-to-cell contacts as much as possible. The cells should be handled gently. Completely confluent wells should be passaged at a ratio of 1:2, while subconfluent wells should be passaged at a ratio of 1:1 or less.
<ol>
	<li>Remove the medium from the culture and briefly rinse with DPBS.</li>
	<li>Add 1 mL of Versene. Incubate at room temperature for 30 s.</li>
	<li>Remove the Versene and add 1 mL of TrypLE. Incubate at 37 &#176;C in the tissue culture incubator for 3 min.</li>
	<li>Observe the culture using a phase contrast microscope. Gently tap the culture to dislodge cells from the plate. As soon as the large fibroblastic cells have detached from the plate remove them and the TrypLE using a 1 mL pipette. Wash residual TrypLE off the remaining cells by rinsing twice with 2 mL of DPBS.</li>
	<li>Add 1 mL of 1 mg/mL Dispase II to the culture and incubate in the tissue culture incubator for 1 to 2 h or until all cells have detached from the plate. The cells should gradually float away from the plate in clumps.</li>
	<li>Collect the cells using a 1 mL pipette and transfer to 20 mL of DPBS in a 50 mL centrifuge tube. Centrifuge for 5 min at 300 x g at room temperature.</li>
	<li>Remove the supernatant and gently resuspend the cell pellet by trituration with a 1 mL pipette in 1 mL of culture medium. Transfer the cell suspension to either one or two wells of a 6-well plate, to passage at a ratio of either 1:1 or 1:2 respectively.</li>
	<li>Top up the medium in each well to make 2 mL and place the cultures into the tissue culture incubator. Replace the medium with 2 mL of fresh medium twice per week.</li>
</ol>
7. Growth of corneal endothelial cell layers on biomaterial membranes
NOTE: The following procedure describes the steps involved in mounting a membranous biomaterial in a custom-made mounting device&#8212;called a micro-Boyden chamber&#8212;for cell culture. Please refer to our recent publication6 for further information about the device and for purchasing details.
<ol>
	<li>Assemble the upper chamber of the micro-Boyden chamber by placing a red O-ring into its center.</li>
	<li>Use a trephine of 18 mm diameter to punch out a disc from a biomaterial sheet on a polytetrafluoroethylene (PTFE) cutting board. Place this disc over the red O-ring in the micro-Boyden chamber&#39;s upper chamber.</li>
	<li>Screw the lower chamber onto the upper chamber, securing the biomaterial disc in between.</li>
	<li>Soak the assembled micro-Boyden chamber in 70% ethanol for 1 h to sterilize it.</li>
	<li>Completely immerse the assembled micro-Boyden chamber in sterile HBSS for 10 min to remove the ethanol. Repeat this wash step twice. Perform a final wash step for 10 min in unsupplemented culture medium.</li>
	<li>Transfer the sterilized micro-Boyden chamber to culture medium in the well of a 6-well plate ensuring that the upper chamber is uppermost. Adjust the level of culture medium so that it contacts the lower surface of the biomaterial membrane but does not flow into the upper chamber.</li>
	<li>Prepare a suspension of corneal endothelial cells using the procedure outlined in section 6. Sufficient cell density in the suspension should be achieved to allow a seeding density of at least 100,000 cells per cm2 on the membrane in the micro-Boyden chamber. The culture surface area within the micro-Boyden chamber is 0.5 cm2 and it can hold a volume of 100 &#181;L. Therefore, a suspension containing 500,000 cells/mL should be prepared.</li>
	<li>Pipette 100 &#181;L of cell suspension (50,000 cells) onto the membrane in the micro-Boyden chamber. Incubate in the tissue culture incubator for 4 h before topping up the medium to completely submerge the chamber. Incubate the culture for the required period of time, replacing the medium twice per week. 
	NOTE: Once the corneal endothelial cells have attached to the upper surface of the biomaterial membrane the micro-Boyden chamber can be flipped over within the culture well so that the lower chamber is uppermost. More cells can then be added to the chamber to initiate cell cultures on the other surface of the membrane. For example, corneal stromal cells may be readily applied to the alternate surface thus mimicking the relative location of corneal cell types as seen within the posterior cornea.</li>
</ol>

<ol>
	<li>G&#252;ell, J. L., El Husseiny, M. A., Manero, F., Gris, O., Elies, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Historical+Review+and+Update+of+Surgical+Treatment+for+Corneal+Endothelial+Diseases.">Historical Review and Update of Surgical Treatment for Corneal Endothelial Diseases.</a> Ophthalmology and Therapy. 3, 1-15 (2014).</li><li>Tan, D. T. H., Dart, J. K. G., Holland, E. J., Kinoshita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+transplantation.">Corneal transplantation.</a> The Lancet. 379 (9827), 1749-1761 (2012).</li><li>Soh, Y. Q., Peh, G. S. L., Mehta, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+issues+for+human+corneal+endothelial+tissue+engineering.">Translational issues for human corneal endothelial tissue engineering.</a> Journal of Tissue Engineering and Regnerative Medicine. 11 (9), 2425-2442 (2017).</li><li>Senoo, T., Joyce, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Cycle+Kinetics+in+Corneal+Endothelium+from+Old+and+Young+Donors.">Cell Cycle Kinetics in Corneal Endothelium from Old and Young Donors.</a> Investigative Ophthalmology, Visual Science. 41 (3), 660-667 (2000).</li><li>Roy, O., Leclerc, V. B., Bourget, J. M., Th&#233;riault, M., Proulx, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+process+of+corneal+endothelial+morphological+change+in+vitro.">Understanding the process of corneal endothelial morphological change in vitro.</a> Investigative Ophthalmology, Visual Science. 56, 1228-1237 (2015).</li><li>Harkin, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mounting+of+Biomaterials+for+Use+in+Ophthalmic+Cell+Therapies.">Mounting of Biomaterials for Use in Ophthalmic Cell Therapies.</a> Cell Transplantation. 26 (11), 1717-1732 (2017).</li><li>Trepat, X., Chen, Z., Jacobson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration.">Cell migration.</a> Comprehensive Physiology. 2 (4), 2369-2392 (2012).</li><li>Walshe, J., Harkin, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+explant+culture+provides+novel+insights+into+the+potential+location+and+phenotype+of+corneal+endothelial+progenitor+cells.">Serial explant culture provides novel insights into the potential location and phenotype of corneal endothelial progenitor cells.</a> Experimental Eye Research. 127, 9-13 (2014).</li><li>Al Abdulsalam, N. K., Barnett, N. L., Harkin, D. G., Walshe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+of+corneal+endothelial+cells+from+sheep.">Cultivation of corneal endothelial cells from sheep.</a> Experimental Eye Research. 173, 24-31 (2018).</li><li>Parekh, M., Ferrari, S., Sheridan, C., Kaye, S., Ahmad, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+Review:+An+Update+on+the+Culture+of+Human+Corneal+Endothelial+Cells+for+Transplantation.">Concise Review: An Update on the Culture of Human Corneal Endothelial Cells for Transplantation.</a> Stem Cells Translational Medicine. 5 (2), 258-264 (2016).</li><li>Peh, G. S., Toh, K. P., Wu, F. Y., Tan, D. T., Mehta, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+of+human+corneal+endothelial+cells+isolated+from+paired+donor+corneas.">Cultivation of human corneal endothelial cells isolated from paired donor corneas.</a> PLoS One. 6 (12), 28310 (2011).</li></ol>

The authors declare that they have no competing financial interests.

A significant technical challenge associated with establishing and expanding human corneal endothelial cells is preventing EMT from occurring in the cultures. EMT can be triggered in corneal endothelial cells by loss of cell-to-cell contact, yet most cell culture protocols for these cells involve enzymatic dissociation to single cells during isolation and subculture10. Here we present an alternative cell culture protocol for corneal endothelial cells that minimizes the risk of cells losing contact...

The cornea is a transparent tissue that is situated at the front of the eye. It is composed of three major layers: an epithelial layer on the outer surface, a middle stroma layer, and an inner layer called the corneal endothelium. The corneal endothelium is a monolayer of cells that sits on a basement membrane called Descemet&#39;s membrane and it maintains the transparency of the cornea by regulating the amount of fluid that enters the stroma from the underlying aqueous humor. Too much fluid within the stroma causes corneal swelling, opacity and vision loss. The endothelium is therefore vital for maintaining vision.
The corneal endothelium can become dysfunctional for a number of reasons including aging, disease and injury, and the only current treatment is transplant surgery. During this surgery, the endothelium and Descemet&#39;s membrane is removed from the patient&#39;s cornea and replaced with a graft of endothelium and Descemet&#39;s membrane obtained from a donor cornea. Many endothelium grafts also contain a thin layer of stromal tissue to aid handling and attachment to the host cornea1.
Worldwide, the demand for corneal donor tissue for transplant surgeries is greater than the amount that can be supplied by eye banks2. There has therefore been a drive to develop tissue-engineered corneal endothelium transplants that could be used to alleviate this shortfall3. The rationale for this is based on the fact that currently, endothelium from an individual cornea can only be transferred to a single patient, however, if the corneal endothelial cells were first expanded and grown on biomaterial scaffolds in tissue culture, they could be used to treat multiple patients.
Major challenges that need to be addressed before tissue-engineered corneal endothelium transplants become a feasible option for surgeons include: (1) establishing techniques for expanding corneal endothelial cells of high quality and for producing mature and functional corneal endothelial cell layers in vitro, and (2) establishing techniques for growing the cells on biomaterial scaffolds to produce tissue-engineered grafts that are equal to, or better than, the donor cornea-derived grafts that are currently used.
Corneal endothelial cells have a very low proliferative potential in vivo but can be stimulated to divide in vitro4. Nevertheless, they have a strong tendency to undergo in vitro epithelial-to-mesenchymal transition (EMT), which reduces their capacity to form a mature, functional endothelial layer. Known triggers for EMT in corneal endothelial cells include exposure to certain growth factors and loss of cell-to-cell contact5. It is thus almost inevitable that corneal endothelial cell cultures that are enzymatically dissociated during subculture will undergo changes associated with EMT. Here, we present a cell culture method for human or sheep corneal endothelial cells that is designed to minimize disruption of cell-to-cell contacts during isolation, expansion and subculture stages, to reduce the potential for EMT. Furthermore, we demonstrate how tissue-engineered grafts that resemble donor cornea-derived endothelium/Descemet&#39;s membrane/stromal tissue grafts can be produced by growing cultured cell layers on both sides of a biomaterial membrane in a custom-made mounting device.

<table><tbody><tr><td>Attachment factor</td><td>Gibco</td><td>S006100</td><td>A 1X sterile solution containing gelatin that is used to coat tissue culture surfaces. Store at 4 &amp;deg;C.</td></tr><tr><td>Bovine pituitary extract</td><td>Gibco</td><td>13028014</td><td>A single vial contains 25 mg. Freeze in aliquots.</td></tr><tr><td>Calcium chloride</td><td>Merck</td><td>C5670</td><td>Dissolve in HBSS to make a 1 mM stock solution. Filter sterilise.</td></tr><tr><td>Centrifuge tube, 50 ml</td><td>Labtek</td><td>650.550.050</td><td></td></tr><tr><td>Chondroitin sulphate</td><td>LKT Laboratories</td><td>C2960</td><td>This is bovine chondroitin sulphate. Dissolve in HBSS to make a 0.08 g/mL stock solution. Filter sterilise and freeze in aliquots.</td></tr><tr><td>Dispase II</td><td>Gibco</td><td>17105-041</td><td>Dissolve in DPBS to make a 2 mg/mL stock solution. Filter sterilise and freeze in aliquots.</td></tr><tr><td>Ethanol</td><td>Labtek</td><td>EA043</td><td>100% undenatured ethanol should be diluted to 70% in deionised water for sterilising instruments and surfaces.</td></tr><tr><td>Foetal bovine serum</td><td>GE Healthcare Australia Pty Ltd</td><td>SH30084.03</td><td>This is a HyClone brand of foetal bovine serum.</td></tr><tr><td>Coverglass No. 1, &amp;Oslash; 13 mm</td><td>Proscitech</td><td>G401-13</td><td>Place sterilised cover slips into 24-well plates for tissue culture.</td></tr><tr><td>HBSS</td><td>Gibco</td><td>14025-092</td><td>Hank&#039;s balanced salt solution, 1X, containing calcium chloride and magnesium chloride.</td></tr><tr><td>L-ascorbic acid 2-phosphate</td><td>Merck</td><td>A8960</td><td>Dissolve in HBSS to make a 150 mM stock solution. Filter sterilise.</td></tr><tr><td>Micro-Boyden chamber</td><td>CNC Components Pty. Ltd.</td><td>Upper ring: QUT-0002-0006, Base ring: QUT-0002-0007</td><td>Both components are made from polytetrafluoroethelyne (PTFE).</td></tr><tr><td>O-ring for micro-Boyden chamber</td><td>Ludowici Sealing Solutions</td><td>RSB012</td><td>Composed of silicon rubber.</td></tr><tr><td>Opti-MEM 1 (1X) + GlutaMAX-1</td><td>Gibco</td><td>51985-034</td><td>A reduced serum medium containing glutamine.</td></tr><tr><td>DPBS</td><td>Gibco</td><td>14190-144</td><td>Dulbecco&#039;s phosphate buffered saline, 1X, without calcium chloride and magnesium chloride.</td></tr><tr><td>Pen Strep</td><td>Gibco</td><td>15140-122</td><td>A 100X antibiotic solution containing 10,000 Units/mL penicillin and 10,000 &amp;micro;g/mL streptomycin.</td></tr><tr><td>Petri dish</td><td>Sarstedt</td><td>82.14473.001</td><td>Sterile Petri dish, 92 X 16 mm, for tissue dissections.</td></tr><tr><td>Tissue culture plate, 24 well</td><td>Corning Incorporated</td><td>Costar 3524</td><td>A plate containing 24 wells, each with a surface area of 2 cm&lt;sup&gt;2&lt;/sup&gt;.</td></tr><tr><td>Tissue culture plate, 6 well</td><td>Corning Incorporated</td><td>Costar 3516</td><td>A plate containing 6 wells, each with a surface area of 9 cm&lt;sup&gt;2&lt;/sup&gt;.</td></tr><tr><td>TrypLE Select</td><td>Gibco</td><td>12563-011</td><td>A 1X enzyme solution for dissociating cells.</td></tr><tr><td>Versene</td><td>Gibco</td><td>15040-066</td><td>A 1X EDTA solution for dissociating cells.</td></tr><tr><td>Watchmaker forceps</td><td>Labtek</td><td>BWMF4</td><td>Number 4 watchmaker forceps work well for removing strips of endothelium/Descemet&#039;s membrane from corneas.</td></tr></tbody></table>

growth of human and sheep corneal endothelial cell layers on biomaterial membranes

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is deleterious for the cells as it reduces their ability to form a mature and functional layer. Here, we present a method for establishing and subculturing human and sheep corneal endothelial cell cultures that minimizes the loss of cell-to-cell contact. Explants of corneal endothelium/Descemet&#39;s membrane are taken from donor corneas and placed into tissue culture under conditions that allow the cells to collectively migrate onto the culture surface. Once a culture has been established, the explants are transferred to fresh plates to initiate new cultures. Dispase II is used to gently lift clumps of cells off tissue culture plates for subculturing. Corneal endothelial cell cultures that have been established using this protocol are suitable for transferring to biomaterial membranes to produce tissue-engineered cell layers for transplantation in animal trials. A custom-made device for supporting biomaterial membranes during tissue culture is described and an example of a tissue-engineered graft composed of a layer of corneal endothelial cells and a layer of corneal stromal cells on either side of a collagen type I membrane is presented.

The method for isolating and expanding corneal endothelial cells from human or sheep corneas is summarized in Figure 1 and Figure 2. Most explants that are derived from the corneas of 1 to 2-year-old sheep or human donors of less than 30 years of age will attach to Attachment Factor-coated tissue culture plates within a week, however, it is not unusual to find that up to one third of explants fail to attach within this time. These 'floating' explants can be remo...

Watch this Scientific Journal Video about Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes at JoVE.com

Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes

This protocol describes the critical steps required to establish and grow corneal endothelial cell cultures from explants of human or sheep tissue. A method for subculturing corneal endothelial cells on membranous biomaterials is also presented.

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is ...

growth-human-sheep-corneal-endothelial-cell-layers-on-biomaterial

Research

JoVE Journal

Bioengineering

7.7K Views.  Queensland Eye Institute. Dit celcultuurprotocol is ontworpen om te voorkomen dat hoornvlies endotheelcellen epitheliale naar mesenchymale overgang ondergaan tijdens isolatie, expansie en subcultuur. Celculturen worden vastgesteld uit hoornvliesweefsel explants, die kunnen worden hergebruikt om een voortdurende levering van primaire cellen van een donor te garanderen. Begin met het coaten van de putten van een zes-goed weefsel kweekplaat met gehechtheid factor met behulp van de instructies van de fabrikant.Voeg...

Video: Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

Developmental Biology

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

A primary culture of bovine corneal endothelial cells was used to investigate the mechanism of corneal endothelial-mesenchymal transition. Furthermore, a rat corneal endothelium cryoinjury model was used to demonstrate corneal endothelial-mesenchymal transition in vivo.

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

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 ...

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 ...

Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms of retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Cell therapy is one of the most promising therapeutic strategies proposed to cure these diseases, with already encouraging preliminary results in humans. However, the method of preparation of the graft has a significant impact on its functional outcomes in vivo. Indeed, RPE cells grafted as a cell suspension are less functional than the same cells transplanted as a retinal tissue. Herein, we describe a simple and reproducible method to engineer RPE tissue and its preparation for an in vivo implantation. RPE cells derived from human pluripotent stem cells are seeded on a biological support, the human amniotic membrane (hAM). Compared to artificial scaffolds, this support has the advantage of having a basement membrane that is close to the Bruch&#39;s membrane where endogenous RPE cells are attached. However, its manipulation is not easy, and we developed several strategies for its proper culturing and preparation for grafting in vivo.

We describe a method to engineer a retinal tissue composed of retinal pigment epithelial cells derived from human pluripotent stem cells cultured on top of human amniotic membranes and its preparation for grafting in animal models.

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Biology

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 ...

Medicine

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for ...

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...

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 ...

Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes

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