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 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 ...

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

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Bioengineering

7.6K Views.  Queensland Eye Institute. 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.

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

On-Chip Endothelial Inflammatory Phenotyping

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual's level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo. We describe a lab-on-a-chip based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC), which are bound directly to cultured EC monolayers.1 These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-,2 lipid-3, 4 and RAGE-induced5 inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-&alpha;. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.

Microfluidic flow chambers etched by photolithography and fabricated from PDMS are applied to probe functional outcomes associated with EC dysfunction and inflammation. In a representative experiment, the ability of differential shear stress to modulate monocytic cell adhesion to cytokine activated EC monolayers is demonstrated.

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

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 ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.

Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing.

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral ...

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

We present a protocol to perform subtractive patterning of live cell monolayers on a surface. This is achieved by local and selective lysis of adherent cells using a microfluidic probe (MFP). The cell lysate retrieved from local regions can be used for downstream analysis, enabling molecular profiling studies.

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

Surface Engineering of Pancreatic Islets with a Heparinized StarPEG Nanocoating

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and survival post-transplantation. This protocol aims to achieve surface engineering of pancreatic islets using an ultrathin heparin-incorporated starPEG (Hep-PEG) nanocoating. To generate the Hep-PEG nanocoating for pancreatic islet surface engineering, heparin succinimidyl succinate (Heparin-NHS) was first synthesized by modification of its carboxylate groups using N-(3-dimethylamino propyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The Hep-PEG mixture was then formed by crosslinking of the amino end-functionalized eight-armed starPEG (starPEG-(NH2)8) and Heparin-NHS. For islet surface coating, mouse islets were isolated via collagenase digestion and gradient purification using Histopaque. Isolated islets were then treated with ice cold Hep-PEG solution for 10 min to allow covalent binding between NHS and the amine groups of islet cell membrane. Nanocoating with the Hep-PEG incurs minimal alteration to islet size and volume and heparinization of the islets with Hep-PEG may also reduce instant blood-mediated inflammatory reaction during islet transplantation. This "easy-to-adopt" approach is mild enough for surface engineering of living cells without compromising cell viability. Considering that heparin has shown binding affinity to multiple cytokines, the Hep-PEG nanocoating also provides an open platform that enables incorporation of unlimited functional biological mediators and multi-layered surfaces for living cell surface bioengineering.

This protocol aims to achieve surface engineering of pancreatic islets using a heparin-incorporated starPEG nanocoating via pseudo-bioorthogonal chemistry between the N-hydroxysuccinimide groups of the nanocoating and the amine groups of islet cell membrane.

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and ...

A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development and implementation of biomedical devices and tissue engineering constructs. Macrophages, an innate immune cell, are key players in the foreign body reaction because they remain at the implant site for the lifetime of the device, and are commonly studied to gain an understanding of this detrimental host response. Many biomaterials researchers have shown that adsorbed protein layers on implanted materials influence macrophage behavior, and subsequently impact the host response. The methods in this paper describe an in vitro model using adsorbed protein layers containing cellular damage molecules on polymer biomaterial surfaces to assess macrophage responses. An NF-&#1082;B/AP-1 reporter macrophage cell line and the associated colorimetric alkaline phosphatase assay were used as a rapid method to indirectly examine NF-&#1082;B/AP-1 transcription factor activity in response to complex adsorbed protein layers containing blood proteins and damage-associated molecular patterns, as a model of the complex adsorbed protein layers formed on biomaterial surfaces in vivo.

This protocol provides researchers with a rapid, indirect method of measuring TLR-dependent NF-&#1082;B/AP-1 transcription factor activity in a murine macrophage cell line in response to a variety of polymeric surfaces and adsorbed protein layers that model the biomaterial implant microenvironment.

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development ...

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. 
One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. 
Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

Fabrication of a Crystalline Nanocellulose Embedded Agarose Biomaterial Ink for Bone Marrow-Derived Mast Cell Culture

Three-dimensional (3D) bioprinting utilizes hydrogel-based composites (or biomaterial inks) that are deposited in a pattern, forming a substrate onto which cells are deposited. Because many biomaterial inks can be potentially cytotoxic to primary cells, it is necessary to determine the biocompatibility of these hydrogel composites prior to their utilization in costly 3D tissue engineering processes. Some 3D culture methods, including bioprinting, require that cells be embedded into a 3D matrix, making it difficult to extract and analyze the cells for changes in viability and biomarker expression without eliciting mechanical damage. This protocol describes as proof of concept, a method to assess the biocompatibility of a crystalline nanocellulose (CNC) embedded agarose composite, fabricated into a 24-well culture system, with mouse bone marrow-derived mast cells (BMMCs) using flow cytometric assays for cell viability and biomarker expression.
After 18 h of exposure to the CNC/agarose/D-mannitol matrix, BMMC viability was unaltered as measured by propidium iodide (PI) permeability. However, BMMCs cultured on the CNC/agarose/D-mannitol substrate appeared to slightly increase their expression of the high-affinity IgE receptor (Fc&#949;RI) and the stem cell factor receptor (Kit; CD117), although this does not appear to be dependent on the amount of CNC in the bioink composite. The viability of BMMCs was also assessed following a time course exposure to hydrogel scaffolds that were fabricated from a commercial biomaterial ink composed of fibrillar nanocellulose (FNC) and sodium alginate using a 3D extrusion bioprinter. Over a period of 6-48 h, the FNC/alginate substrates did not adversely affect the viability of the BMMCs as determined by flow cytometry and microtiter assays (XTT and lactate dehydrogenase). This protocol describes an efficient method to rapidly screen the biochemical compatibility of candidate biomaterial inks for their utility as 3D scaffolds for post-print seeding with mast cells.

This protocol highlights a method to rapidly assess the biocompatibility of a crystalline nanocellulose (CNC)/agarose composite hydrogel biomaterial ink with mouse bone marrow-derived mast cells in terms of cell viability and phenotypic expression of the cell surface receptors, Kit (CD117) and high-affinity IgE receptor (Fc&#949;RI).

Three-dimensional (3D) bioprinting utilizes hydrogel-based composites (or biomaterial inks) that are deposited in a pattern, forming a substrate onto ...