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 soaki...

<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>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#176;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, &#216; 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&#39;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&#39;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 &#181;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 cm2.</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 cm2.</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&#39;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 &#39;floating&#39; explants can...

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 ...

This cell culture protocol is designed to prevent corneal endothelial cells from undergoing epithelial to mesenchymal transition during isolation, expansion, and subculture. Cell cultures are established from corneal tissue explants, which can be reused to ensure an ongoing supply of primary cells from one donor. Begin by coating the wells of a six-well tissue culture plate with attachment factor using the manufacturer's instructions.Then add one milliliter of culture medium to each coded well. 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, and adjust the zoom so that the edge and some central corneal endothelium is in view.Use sterilized watchmaker forceps to gently lift and tear Descemet's membrane away from the underlying stroma. Place the membrane strip into one of the prepared wells of the culture plate, making sure to immediately replace the lid of the plate to reduce the risk of contamination. Remove the strips of Descemet's membrane from the extreme periphery of the endothelium first, and from the central regions later, placing all strips from one cornea into a single well.When finished, incubate the explants in a humidified cell culture incubator set to 37 degrees Celsius and 5%carbon dioxide. Assemble the upper chamber of the micro Boyden chamber by placing a red O ring into its center. Use an 18 millimeter diameter trephine to punch out a disk from a biomaterial sheet on a PTFE cutting board.Then, place the disk over the red O ring in the micro Boyden chamber's upper chamber. Screw the lower chamber onto the upper chamber, securing the bio material's disk in between. And soak the assembled micro Boyden chamber in 70%ethanol for one hour to sterilize it.After the soak, completely immerse the chamber in sterile HBSS for 10 minutes to remove the ethanol. Then wash the chamber in culture medium for 10 minutes. And transfer to culture medium inside a well of a six-well plate, making sure that the upper chamber is oriented upwards.Adjust the level of the medium so that it contacts the lower surface of the biomaterial membrane, but does not flow into the upper chamber. Pipette 100 microliters of cell suspension onto the membrane into the micro Boyden chamber, and incubate it in the tissue culture incubator for four hours, before adding medium to completely submerge the chamber. Most explants that were derived from the corneas of one to two-year-old sheep or human donors of less than 30 years of age attached to attachment factor coded tissue culture plates within a week.After six weeks, many small, tightly-packed cells were present next to the explant. Small, tightly-packed cells within the corneal endothelial cell cultures are resistant to digestion with triple, while the larger fibroblastic cells are more sensitive to it. This difference was exploited to selectively remove large cells from the cultures before transferring the smaller cells to new plates.Immunal flourescence analyses were conducted on corneal endothelial cell cultures to locate ZO-1, a tight junction protein, and N-cadherin, an adherence junction protein. The same antibodies were used for both sheep and human cells, and both proteins were detected in the plasma membranes of cells from both species. The custom-made micro Boyden chamber allowed both sides of a membrane to be used as cell culture surfaces simultaneously.A cross-section view of a tissue engineered cell construct shows a single layer of corneal endothelial cells on one surface, and a multilayered culture of corneal stromal cells on the other. When attempting this protocol, it's important to avoid scraping the endothelium with your forceps when you're peeling Descemet's membrane away from the cornea. Following this procedure, the integrity of a corneal endothelial cell layer on a membrane can be checked by measuring its electrical resistance.

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Bioengineering

7.6K visualizações.  Queensland Eye Institute. Este protocolo de cultura celular foi projetado para evitar que as células endoteliais da córnea submetam a transição epitelial para mesenquimal durante o isolamento, expansão e subcultura. Culturas celulares são estabelecidas a partir de explanações de tecido córnea, que podem ser reutilizadas para garantir um fornecimento contínuo de células primárias de um doador. Comece revestindo os poços de uma placa de cultura de tecido de seis poços com fator de fixação usando as instr...