We would like to acknowledge the NIH for the following grant support: RO1EY032079 (VTR), R21EY029097-01 (VTR), 1F30EY033647-01 (KS), and 5T32GM008541-24 (KS). We would also like to acknowledge the Massachusetts Lions Eye Research Fund and the New England Corneal Transplant Fund.

The procedures involving animal subjects were approved by the Association for Research in Vision and Ophthalmology for the Use of Animals in Ophthalmic Care and Vision Research and the Boston University IACUC protocol (201800302).
1. Designing the 3D printed holders and cover bar
<ol>
	<li>Design the 3D printed holders and cover bar accounting for the average diameter of mouse globes and 3D print the design (Figure 1A, B).</li>
...

<ol>
	<li>Kneer, K., 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=High+fat+diet+induces+pre-type+2+diabetes+with+regional+changes+in+corneal+sensory+nerves+and+altered+P2X7+expression+and+localization.">High fat diet induces pre-type 2 diabetes with regional changes in corneal sensory nerves and altered P2X7 expression and localization.</a> Experimental Eye Research. 175, 44-55 (2018).</li><li>Lee, Y., 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=Sustained+Ca2++mobilizations:+A+quantitative+approach+to+predict+their+importance+in+cell-cell+communication+and+wound+healing.">Sustained Ca2+ mobilizations: A quantitative approach to predict their importance in cell-cell communication and wound healing.</a> PLoS One. 14 (4), 0213422 (2019).</li><li>Stepp, M. A., 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=Wounding+the+cornea+to+learn+how+it+heals.">Wounding the cornea to learn how it heals.</a> Experimental Eye Research. 121, 178-193 (2014).</li><li>Klepeis, V. S., Cornell-Bell, A., Trinkaus-Randal, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+factors+but+not+gap+junctions+play+a+role+in+injury-induced+Ca2++waves+in+epithelial+cells.">Growth factors but not gap junctions play a role in injury-induced Ca2+ waves in epithelial cells.</a> Journal of Cell Science. 114 (23), 4185-4195 (2001).</li><li>Lee, A., 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=Hypoxia-induced+changes+in+Ca(2+)+mobilization+and+protein+phosphorylation+implicated+in+impaired+wound+healing.">Hypoxia-induced changes in Ca(2+) mobilization and protein phosphorylation implicated in impaired wound healing.</a> American Journal of Physiology. Cell Physiology. 306 (10), 972-985 (2014).</li><li>Boucher, I., Rich, C., Lee, A., Marcincin, A., Trinkaus-Randall, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+P2Y2+receptor+mediates+the+epithelial+injury+response+and+cell+migration.">The P2Y2 receptor mediates the epithelial injury response and cell migration.</a> American Journal of Physiology. Cell Physiology. 299 (2), 411-421 (2010).</li><li>Awal, M. R., Wirak, G. S., Gabel, C. V., Connor, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collapse+of+global+neuronal+states+in+Caenorhabditis+elegans+under+isoflurane+anesthesia.">Collapse of global neuronal states in Caenorhabditis elegans under isoflurane anesthesia.</a> Anesthesiology. 133 (1), 133-144 (2020).</li><li>Burnett, K., Edsinger, E., Albrecht, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+gentle+hydrogel+encapsulation+of+living+organisms+enables+long-term+microscopy+over+multiple+hours.">Rapid and gentle hydrogel encapsulation of living organisms enables long-term microscopy over multiple hours.</a> Communications Biology. 1, 73 (2018).</li><li>Rhodes, 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=Pannexin1:+Role+as+a+sensor+to+injury+is+attenuated+in+pretype+2+corneal+diabetic+epithelium.">Pannexin1: Role as a sensor to injury is attenuated in pretype 2 corneal diabetic epithelium.</a> Analytical Cellular Pathology. 2021, 4793338 (2021).</li><li>Segars, K. L., 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=Age+dependent+changes+in+corneal+epithelial+cell+signaling.">Age dependent changes in corneal epithelial cell signaling.</a> Frontiers in Cell and Developmental Biology. 10, 886721 (2022).</li><li>Xu, P., Londregan, A., Rich, C., Trinkaus-Randall, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+epithelial+and+stromal+corneal+stiffness+occur+with+age+and+obesity.">Changes in epithelial and stromal corneal stiffness occur with age and obesity.</a> Bioengineering. 7 (1), 14 (2020).</li><li>Minns, M. S., Teicher, G., Rich, C. B., Trinkaus-Randall, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purinoreceptor+P2X7+regulation+of+Ca(2+)+mobilization+and+cytoskeletal+rearrangement+is+required+for+corneal+reepithelialization+after+injury.">Purinoreceptor P2X7 regulation of Ca(2+) mobilization and cytoskeletal rearrangement is required for corneal reepithelialization after injury.</a> The American Journal of Pathology. 186 (2), 285-296 (2016).</li><li>Tadvalkar, G., Pal-Ghosh, S., Pajoohesh-Ganji, A., Stepp, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+euthanasia+and+enucleation+on+mouse+corneal+epithelial+axon+density+and+nerve+terminal+morphology.">The impact of euthanasia and enucleation on mouse corneal epithelial axon density and nerve terminal morphology.</a> The Ocular Surface. 18 (4), 821-828 (2020).</li></ol>

This protocol describes a&#160;live cell imaging technique that uses a 3D printed holder to stabilize and immobilize intact animal eyes. It is designed to circumvent several significant disadvantages recognized with previous live cell imaging protocols of ex vivo corneal tissue. This protocol offers many advantages for the live cell imaging of intact globes. It significantly reduces unnecessary tissue damage that could interfere with the wound healing response to experimentally induced scratch wounds. This inclu...

Cornea 
The cornea is a clear, avascular structure covering the anterior surface of the eye that refracts light to enable vision and protects the interior of the eye from damage. As the cornea is exposed to the environment, it is susceptible to damage from both mechanical causes (scratching) and from infection. A corneal injury in an otherwise healthy patient typically heals within 1-3 days. However, in patients with underlying conditions including limbal stem cell deficiency and type II diabetes, the corneal wound healing process can be greatly prolonged1. As the cornea is highly innervated, these non-healing corneal ulcers and recurrent corneal erosions are very painful and greatly diminish the quality of life of patients experiencing them1.
Cell signaling 
When an otherwise healthy cornea is injured, calcium signaling events in the cells adjacent to the wound precede and prompt cellular migration into the wound bed, where they close the injury without the risk of scarring2,3. These signaling events have been well-characterized in corneal epithelial cell culture models using live cell imaging2. Preliminary experiments demonstrate significantly more calcium signaling after injury in non-diabetic cells compared to diabetic cells. However, characterization of the cell signaling events in ex vivo globes has proved to be a technical challenge.
Live cell imaging 
Previous studies have successfully recorded calcium signaling events from in vitro cell culture models of corneal wound healing4,5,6. Developing a methodology to produce high-quality images of these signaling events in ex vivo tissue is of great interest because it would permit the study of these events in a more complex and true-to-life system. Previous approaches have involved dissection of the cornea followed by immobilization in a UV-induced PEG gel7,8,9. Immobilization is an essential yet challenging step when working with live tissue, as it must remain viable and hydrated throughout the course of the experiment. Furthermore, immobilization must not damage the tissue. While the PEG solution immobilized the tissue, the resolution and quality of the images produced were not consistent. Therefore, 3D printed holders were developed to immobilize intact globes to produce higher-quality images with less risk of tissue damage.
The approach 
A unique 3D printed holder was developed to immobilize intact ex vivo globes for live cell imaging. This holder prevents damage from two major sources: it allows for imaging of an enucleated globe without the need to dissect the cornea, and it eliminates exposure to UV light. Without these sources of damage, the images obtained more accurately represented the response to the scratch injuries made experimentally. Furthermore, the 3D printed holder was calibrated to the precise dimensions of the murine eye. This provided a much better fit than immobilization in PEG solution, leading to a higher-quality image at lower-powered objectives due to decreased tissue movement. A cover bar attached to the top of the holder ensures that the globe remains immobile throughout the duration of the experiment and that there is no displacement of the globe when growth media is applied to maintain hydration and viability. The ability to print the holder to precise dimensions also allows us to generate an optimal fit for murine eyes of different sizes due to the age or disease status. This technology can be applied more broadly to develop holders for the eyes of different species based on their dimensions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.75 blue polylactic acid (PLA) plastic</td>
			<td>Creality (Shenzen, China)</td>
			<td>N/A</td>
			<td>Material for holder</td>
		</tr>
		<tr>
			<td>35 mm Dish, No. 1.5 Coverslip, 14 mm glass diameter, Poly-D-Lysine Coated</td>
			<td>MatTek Corporation (Ashland, MA)</td>
			<td>P35GC-1.5-14-C</td>
			<td>Well for imaging.&#160;</td>
		</tr>
		<tr>
			<td>Autodesk Fusion 360 software</td>
			<td>Autodesk (San Rafael, CA).</td>
			<td>N/A</td>
			<td>Software used for printing the holders.</td>
		</tr>
		<tr>
			<td>BD 25 G 7/8 sterile needles single use 100 needles/box</td>
			<td>Thermo Fisher Scientific (Waltham, MA)</td>
			<td>305124</td>
			<td>For experimentally-induced wounds to the globes</td>
		</tr>
		<tr>
			<td>CellMask DeepRed</td>
			<td>Invitrogen (Carlsbad, CA)</td>
			<td>C10046</td>
			<td>Cell membrane counterstain. Calcium indicator. 1:10,000 concentration with a final concentration of 1%(v/v) DMSO and 0.1% (w/v) pluronic acid</td>
		</tr>
		<tr>
			<td>Complete Home Super Glue</td>
			<td>Walgreens (Deerfield, IL)</td>
			<td>N/A</td>
			<td>For attaching the holder to the imaging well</td>
		</tr>
		<tr>
			<td>Ender 3 Pro 3D printer&#160;</td>
			<td>Creality (Shenzen, China)</td>
			<td>N/A</td>
			<td>For printing the holder</td>
		</tr>
		<tr>
			<td>FIJI/ImageJ</td>
			<td>ImageJ (Bethesda, MD)</td>
			<td>License Number: GPL2</td>
			<td>Softwareused for confirming consistency of wound depth and diameter between independent globes using Region of Interest analysis</td>
		</tr>
		<tr>
			<td>Fluo-4</td>
			<td>Invitrogen (Carlsbad, CA)</td>
			<td>F14201</td>
			<td>Calcium indicator. 1:100 concentration with a final concentration of 1%(v/v) DMSO and 0.1% (w/v) pluronic acid</td>
		</tr>
		<tr>
			<td>Keratinocyte Serum-Free Medium</td>
			<td>Gibco (Waltham, MA)</td>
			<td>17005042</td>
			<td>25 mg/mL bovine pituitary extract, 0.02 nM EGF, 0.3 mM CaCl2, and penicillin-streptomycin (100 units/mL, 100 &#181;g/mL, respectively) added to medium</td>
		</tr>
		<tr>
			<td>Phophate-Buffered Saline (PBS)</td>
			<td>Corning, Medlabtech (Manassas, VA)</td>
			<td>21-040-CV</td>
			<td>Used to wash excess stain off of corneas before imaging</td>
		</tr>
		<tr>
			<td>Zeiss Confocal 880 Microscope with AiryScan</td>
			<td>Zeiss (Thornwood, NY)</td>
			<td>N/A</td>
			<td>20x magnification objective was used</td>
		</tr>
	</tbody>
</table>

live-cell imaging of intact ex vivo globes using a novel 3d printed holder

Corneal epithelial wound healing is a migratory process initiated by the activation of purinergic receptors expressed on epithelial cells. This activation results in calcium mobilization events that propagate from cell to cell, which are essential for initiating cellular motility into the wound bed, promoting efficient wound healing. The Trinkaus-Randall lab has developed a methodology for imaging the corneal wound healing response in ex vivo murine globes in real time. This approach involves enucleating an intact globe from a mouse that has been euthanized per established protocols and immediately incubating the globe with a calcium indicator dye. A counterstain that stains other features of the cell can be applied at this stage to assist with imaging and show cellular landmarks. The protocol worked well with several different live cell dyes used for counterstaining, including SiR actin to stain actin and deep red plasma membrane stain to stain the cell membrane. To examine the response to a wound, the corneal epithelium is injured using a 25 G needle, and the globes are placed in a 3D printed holder. The dimensions of the 3D printed holder are calibrated to ensure immobilization of the globe throughout the duration of the experiment and can be modified to accommodate eyes of different sizes. Live cell imaging of the wound response is performed continuously at various depths throughout the tissue over time using confocal microscopy. This protocol allows us to generate high-resolution, publication-quality images using a 20x air objective on a confocal microscope. Other objectives can also be used for this protocol. It represents a significant improvement in the quality of live cell imaging in ex vivo murine globes and permits the identification of nerves and epithelium.

This protocol has been used to consistently produce publication-quality data and images10. The images obtained represent a significant improvement when compared to previous approaches (Figure 2). Using the 3D printed holder, images can be captured throughout the layers of the cornea, and calcium mobilization in different z-planes can be observed (Figure 3). This approach has been used to compare cell-cell signaling between apical and basa...

Watch this Scientific Journal Video about Live-Cell Imaging of Intact Ex Vivo Globes Using a Novel 3D Printed Holder at JoVE.com

Live-Cell Imaging of Intact Ex Vivo Globes Using a Novel 3D Printed Holder

The present work describes a novel experimental protocol that utilizes a 3D printed holder to enable high-resolution live cell imaging of enucleated globes. Through this protocol, the cellular calcium signaling activity in wounded corneal epithelium from ex vivo globes can be observed in real time.

Live-Cell Imaging of Intact Ex Vivo Globes Using a Novel 3D Printed Holder

Corneal epithelial wound healing is a migratory process initiated by the activation of purinergic receptors expressed on epithelial cells. This activation ...

The 3D printed holder used in this protocol allows us to monitor short-term cell signaling and long-term cellular migration events in wounded corneas in real time. By keeping the globe intact, viable, and immobile, this protocol produces high quality live images of corneal cells, including the epithelium and nerves. The 3D printed holder could easily be modified for globes of various sizes and species.We can also orient the eyes differently to image other regions or perform nano indention experiments. To begin, after removing the head of a euthanized mouse, place it immediately on ice to preserve the viability of the tissue. Then enucleate the globes by prop dozing it, using tweezers.And clip the optic nerve using dissection scissors just below where it is held by the tweezers. Incubate the globes in two milliliters of the medium in a P-35 cell culture dish, including a calcium indicator and/or cell membrane stain for one hour in an incubator at 37 degrees Celsius and 5%carbon dioxide with low light conditions, ensuring that the globes are submerged in the stain medium for uniform staining. To adhere the holders to a clean glass bottom cover slip, wash the holder in 70%ethanol and place glue onto the holder.Then, adhere the holder to the glass bottom cover slip, ensuring no glue is within the inner area of the holder, as glue can fluoresce, complicating imaging. After the glue solidifies, confirm that the holder is secure against the cover slip. Remove the globes from the staining solution using the sterile eyedroppers, taking care to prevent tissue damage to the region of interest.Wash the globes for five minutes at room temperature, using sterile phosphate buffered saline to remove excess stain and place the globes in the medium for transport to the microscope. To wound the globes using a sterile 25 gauge needle in the region of interest, pick up and hold the globe from the back of the eye, using a sterile eyedropper to keep the globe stable and prevent it from rolling. For a scratch wound, gently move a sterile 25 gauge needle across the exposed cornea or gently press the needle directly into the central cornea for a puncture wound, ensuring the wound does not pierce the corneal basement membrane.Place the cornea or limbal region onto the cover slip in the inner area of the holder, while confirming the globe is positioned correctly and that the site of interest is in contact with the glass cover slip. Stabilize using the 3D printed cover adhered to the holder, using glue. And do not try to remove the globe, as this may cause tissue damage.Turn on the microscope and set the environmental chamber to 35 degrees Celsius and 5%carbon dioxide and verify the humidified chamber. Place the cover slip holder and stabilized globe on the microscope stage within the environmental chamber and image, using live cell imaging techniques. By vet additional growth media onto the cover slip to prevent dehydration and maintain tissue viability, ensuring there is enough medium in the well to cover the globe in the holder.Begin experiments using live cell imaging techniques, while using low power laser settings to preserve the tissue and prevent tissue damage and appropriate objectives for long duration experiments. Record and save data in the preferred file format of CZI files for data recording. High quality imaging data of multi-layered structure was obtained for X vivo cornea, stabilized with a 3D printed holder, demonstrating calcium signaling events in the apical, basal, and stromal layers of the cornea.Z-stack images of a cornea, immobilized in the 3D printed holder, at a scratch wound were obtained to visualize the cell membranes and calcium signaling throughout the layers of the cornea and calcium mobilization in different Z planes. A time series experiment of a cornea was performed to study the cellular migration into the wound bed during the healing response, revealing little movement of the globe in the X, Y, or Z directions. It was observed that by placing the globe in the holder, different regions of the cornea, both at the central and in the corneal limbal region can be visualized.This protocol allows us to image eyes from disease models that exhibit differences in wound healing. We can characterize aberrations in cell signaling and motility in live tissue.

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Research

JoVE Journal

Biochemistry

2.5K visualizações.  Boston University. O suporte impresso em 3D usado neste protocolo nos permite monitorar a sinalização celular de curto prazo e os eventos de migração celular de longo prazo em córneas feridas em tempo real. Ao manter o globo intacto, viável e imóvel, este protocolo produz imagens vivas de alta qualidade das células da córnea, incluindo o epitélio e os nervos. O suporte impresso em 3D poderia ser facilmente modificado para globos de vários tamanhos e espécies.Também podemos orientar os olhos ...