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>
	<li>Keep the diameter of the inner wall of the holder slightly larger than the average diameter of the globe to account for the different sizes of individual mice. Keep the height of the holder at around half of the globe diameter, ensuring a tight fit of the globe when secured by the 3D printed cover bar.</li>
	<li>Size the holder cover bar to the length of the holder&#39;s outer diameter with a width that is 1/4 to 1/2 of the holder diameter. The cover bar is sized to allow for access to the globe when secured in the holder for hydration and the removal of the eye at the conclusion of the experiment.</li>
	<li>Print the holder and cover bar.</li>
</ol>
2. Sample collection
<ol>
	<li>Euthanize mice (male C57BL/6 mice aged 9-12 weeks and 27 weeks old were used for this study) using established protocols in compliance with institutional guidelines. For this protocol, perform euthanasia with carbon dioxide followed by decapitation.</li>
	<li>Remove the mouse head and place it immediately on ice to preserve the viability of the tissue. Enucleate the globes using dissection tools while preventing tissue damage.</li>
	<li>Proptose the globe using tweezers. Clip the optic nerve using dissection scissors just below where it is held by the tweezers. 
	NOTE: For further precautions, perform the following steps in a laminar flow hood.</li>
	<li>Incubate the globes in 2 mL of medium in a p35 cell culture dish including a calcium indicator and/or cell membrane stain for 1 h in a 37 &#176;C, 5% CO2 incubator with low light conditions. Ensure the globes are submerged in the staining medium for uniform staining.
	<ol>
		<li>For the experiments performed here, use the calcium indicator, Fluo4-AM (1:100)2, and cell membrane counter stain, deep red plasma membrane stain (1:10,000)2, with a final concentration of 1% (v/v) DMSO and 0.1% (w/v) pluronic acid in 2 mL of keratinocyte serum-free medium (KSFM) with the following growth supplements: 25 &#181;g/mL bovine pituitary extract, 0.02 nM epidermal growth factor, 0.3 mM CaCl2, and penicillin-streptomycin (100 units/mL and 100 &#181;g/mL, respectively). 
		&#8203;NOTE: The incubation conditions and times are variable depending on the calcium indicator, tissue type, and sample volume. When using pluronic acid, caution is advised as it renders tissue permeable. This protocol calls for 10% pluronic acid. Lower concentrations of pluronic acid have been determined experimentally to be ineffective, and higher concentrations risk damage to the tissue.</li>
	</ol>
	</li>
</ol>
3. Preparation of sample holders
<ol>
	<li>Adhere the holders to a clean glass-bottom coverslip with glue that has not been used previously. The glue used in this protocol comes from single-use containers to ensure sterility and a new, unopened container is used every time.</li>
	<li>Wash the holder in 70% ethanol. Place glue onto the holder and adhere the holder to the glass bottom coverslip. Ensure no glue is within the inner area of the holder as glue can fluoresce, complicating imaging.</li>
	<li>Wait until the glue solidifies. Confirm the holder is secure against the coverslip. 
	NOTE: P35 cell plates with glass-bottom coverslips were used for the experiments presented in this manuscript. Other glass-bottom slides and/or plates can be substituted based on the needs of the experiment.</li>
</ol>
4. Wounding of the eye globes
<ol>
	<li>Remove the globes from the staining solution using sterile eye droppers, taking care to prevent tissue damage to the region of interest. Wash the globes for 5 min at room temperature using sterile phosphate-buffered saline to remove excess stain, and place the globes in the medium for transport to the microscope.</li>
	<li>Wound the globes using a sterile 25 G needle in the region of interest.
	<ol>
		<li>Using a sterile eye dropper, pick up and hold the globe from the back of the eye. This will keep the globe stable, prevent it from rolling, and allow consistent wounds to be made. Using this setup, the optic nerve will be inside the eye dropper nozzle, and the cornea will be facing outward.</li>
		<li>For a scratch wound, gently move a sterile 25 G needle across the exposed cornea. For a puncture wound, gently press the needle directly into the central cornea. Ensure the wound does not puncture the cornea. 
		&#8203;NOTE: Skip this step if a wound response or wounded environment is not required for the experiment. Previous studies have shown that both scratch wounds and puncture wounds to murine corneas made using this method are consistent in both diameter and depth10. Confirmation of the wound dimensions between independent globes was performed using a region of interest analysis.</li>
	</ol>
	</li>
</ol>
5. Sample placement on the holder
<ol>
	<li>Place the cornea or limbal region onto the coverslip in the inner area of the holder and stabilize using the 3D printed cover (Figure 1C-H).</li>
	<li>Confirm that the globe is positioned correctly and that the site of interest is in contact with the glass coverslip. Once the globe has been placed into the holder, do not try to remove the globe as this may cause tissue damage.</li>
	<li>Adhere the 3D printed cover to the holder using glue, ensuring stabilization. Ensure the cover bar adheres to the holder and not the globe. 
	&#8203;NOTE: The area to be imaged is placed down because the protocol is written for use on an inverted microscope. The protocol can be adapted for upright microscopes using holders with a smaller inner radius and the removal of the cover bar. This will result in less globe stabilization.</li>
</ol>
6. Sample imaging
<ol>
	<li>Turn on the microscope and environmental chamber and verify that the chamber is humidified. Set the environmental chamber to 35 &#176;C and 5% CO2 for the duration of the experiment. 
	NOTE: Microscopes with environmental chambers are preferable for this procedure to prevent dehydration and to keep the globe at optimal temperatures but are not required.</li>
	<li>Place the coverslip, holder, and stabilized globe on the microscope stage within the environmental chamber and image using live cell imaging techniques9.</li>
	<li>Pipette additional growth media onto the coverslip to prevent dehydration and maintain tissue viability. Ensure there is enough medium in the well to cover the globe in the holder. Depending on the duration of imaging, add fresh medium when needed throughout the experiment.</li>
	<li>Begin experiments using live cell imaging techniques and protocols. Use low power laser settings to preserve the tissue and prevent tissue damage in long-duration experiments. Use appropriate objectives for long working distances. The experiments in this manuscript were performed using a 20x objective. 
	NOTE: The laser power and gain, experimental duration, location, and plane of imaging are all variables depending on the experimental parameters. Imaging experiments on intact globes ranging from 1 h to 4 h in duration have been performed successfully in past publications10.</li>
	<li>Record and save data in the preferred file format. The software used by the microscope produces .czi files for data recording.</li>
	<li>Dispose of the globes as per the standard institutional protocols at the end of the protocol.</li>
</ol>

<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>

We have no conflicts of interest to disclose.

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><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.&amp;nbsp;</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&amp;nbsp;</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 CaCl&lt;sub&gt;2&lt;/sub&gt;, and penicillin-streptomycin (100 units/mL, 100 &amp;micro;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 ...

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

Research

JoVE Journal

Biochemistry

2.5K Views.  Boston University. De 3D-geprinte houder die in dit protocol wordt gebruikt, stelt ons in staat om op korte termijn celsignalering en cellulaire migratiegebeurtenissen op lange termijn in gewonde hoornvliezen in realtime te volgen. Door de wereldbol intact, levensvatbaar en onbeweeglijk te houden, produceert dit protocol live beelden van hoge kwaliteit van hoornvliescellen, inclusief het epitheel en de zenuwen. De 3D-geprinte houder kan eenvoudig worden aangepast voor globes van verschillende groottes en soorte...

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

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

Bioengineering

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids represent useful models to study the mechanisms that control stem cell self-renewal and differentiation in mammals, including primary ciliogenesis and ciliary signaling. Primary ciliogenesis is the dynamic process of assembling the primary cilium, a key cell signaling center that controls stem cell self-renewal and/or differentiation in various tissues. Here we present a comprehensive protocol for the immunofluorescence staining of cell lineage and primary cilia markers, in whole-mount mouse mammary organoids, for light sheet microscopy. We describe the microscopy imaging method and an image processing technique for the quantitative analysis of primary cilium assembly and length in organoids. This protocol enables a precise analysis of primary cilia in complex three-dimensional structures at the single cell level. This method is applicable for immunofluorescence staining and imaging of primary cilia and ciliary signaling in mammary organoids derived from normal and genetically modified stem cells, from healthy and pathological tissues, to study the biology of the primary cilium in health and disease.

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

Stem cell-derived organoids facilitate the analysis of molecular and cellular processes that regulate stem cell self-renewal and differentiation during organogenesis in mammalian tissues. Here we present a protocol for the analysis of the biology of the primary cilium in mouse mammary organoids.

Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids ...

Genetics

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Biology

Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its restrictions. Producing few repeatable results of pattern formation deteriorates the analysis of the mechanisms underlying the self-organization. Reducing variation in initial culture conditions, such as the cell density and distribution in the extracellular matrix (ECM), is crucial to enhance the repeatability of a 3D culture. In this article, we demonstrate a simple but robust procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain highly repeatable pattern formations. A micromold with a desired shape was fabricated by using photolithography or a machining process, and it formed a 3D pocket in the ECM contained in a hybrid gel cube (HGC). Highly concentrated cells were then injected in the pocket so that the cell cluster shape matched with the fabricated mold shape. The employed HGC allowed multi-directional scanning by its rotation, which enabled high-resolution imaging and the capture of the entire tissue structure even though a low-magnification lens was used. Normal human bronchial epithelial cells were used to demonstrate the methodology.&#160;

We present a procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain a repeatable pattern formation. A cubic device containing two different hydrogels is employed to achieve multi-directional imaging for tissue pattern formation.

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Using Chick Embryo Brain as a Model to Study Human Glioblastoma Cell Behavior

Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior

The chick embryo has been an ideal model system for the study of vertebrate development, particularly for experimental manipulations. Use of the chick embryo has been extended for studying the formation of human glioblastoma (GBM) brain tumors in vivo and the invasiveness of tumor cells into surrounding brain tissue. GBM tumors can be formed by injection of a suspension of fluorescently labeled cells into the E5 midbrain (optic tectum) ventricle in ovo. 
Depending on the GBM cells, compact tumors randomly form in the ventricle and within the brain wall, and groups of cells invade the brain wall tissue. Thick tissue sections (350 &#181;m) of fixed E15 tecta with tumors can be immunostained to reveal that invading cells often migrate along blood vessels when analyzed by 3D reconstruction of confocal z-stack images. Live E15 midbrain and forebrain slices (250-350 &#181;m) can be cultured on membrane inserts, where fluorescently labeled GBM cells can be introduced into non-random locations to provide ex vivo co-cultures to analyze cell invasion, which also can occur along blood vessels, over a period of about 1 week. These ex vivo co-cultures can be monitored by widefield or confocal fluorescence time-lapse microscopy to observe live cell behavior. 
Co-cultured slices then can be fixed, immunostained, and analyzed by confocal microscopy to determine whether or not the invasion occurred along blood vessels or axons. Additionally, the co-culture system can be used for investigating potential cell-cell interactions by placing aggregates of different cell types and colors in different precise locations and observing cell movements. Drug treatments can be performed on ex vivo cultures, whereas these treatments are not compatible with the in ovo system. These two complementary approaches allow for detailed and precise analyses of human GBM cell behavior and tumor formation in a highly manipulatable vertebrate brain environment.

Author Spotlight: Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior  

Chick embryos are used for studying human glioblastoma (GBM) brain tumors in ovo and in ex vivo brain slice co-cultures. GBM cell behavior can be recorded by time-lapse microscopy in ex vivo co-cultures, and both preparations can be analyzed at the experimental endpoint by detailed 3D confocal analysis.

The chick embryo has been an ideal model system for the study of vertebrate development, particularly for experimental manipulations. Use of the chick ...

Neuroscience

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...

Time-Resolved Fluorescence Imaging and Analysis of Cancer Cell Invasion in the 3D Spheroid Model

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major clinical challenge because no efficient method exist for their elimination once their dissemination is underway. A better understanding of the mechanisms regulating cancer cell invasion may lead to the development of novel potent therapies. Due to their physiological resemblance to tumors, spheroids embedded in collagen I have been extensively utilized by researchers to study the mechanisms governing cancer cell invasion into the extracellular matrix (ECM). However, this assay is limited by (1) a lack of control over the embedding of spheroids into the ECM; (2) high cost of collagen I and glass bottom dishes, (3) unreliable immunofluorescent labeling, due to the inefficient penetration of antibodies and fluorescent dyes and (4) time-consuming image processing and quantification of the data. To address these challenges, we optimized the three-dimensional (3D) spheroid protocol to image fluorescently labeled cancer cells embedded in collagen I, either using time-lapse videos or longitudinal imaging, and analyze cancer cell invasion. First, we describe the fabrication of a spheroid imaging device (SID) to embed spheroids reliably and in a minimal collagen I volume, reducing the assay cost. Next, we delineate the steps for robust fluorescence labeling of live and fixed spheroids. Finally, we offer an easy-to-use Fiji macro for image processing and data quantification. Altogether, this simple methodology provides a reliable and affordable platform to monitor cancer cell invasion in collagen I. Furthermore, this protocol can be easily modified to fit the users&#8217; needs.

Presented here is a protocol for the fabrication of a spheroid imaging device. This device enables dynamic or longitudinal fluorescence imaging of cancer cell spheroids. The protocol also offers a simple image processing procedure for the analysis of cancer cell invasion.

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major ...

A High-Throughput Platform for Culture and 3D Imaging of Organoids

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by standard imaging approaches. This protocol describes a way to prepare microfabricated organoid culture chips, which enable multiscale, 3D live imaging on a user-friendly instrument requiring minimal manipulations and capable of up to 300 organoids/h imaging throughput. These culture chips are compatible with both air and immersion objectives (air, water, oil, and silicone) and a wide range of common microscopes (e.g., spinning disk, point scanner confocal, wide field, and brightfield). Moreover, they can be used with light-sheet modalities such as the single-objective, single-plane illumination microscopy (SPIM) technology (soSPIM). 
The protocol described here gives detailed steps for the preparation of the microfabricated culture chips and the culture and staining of organoids. Only a short length of time is required to become familiar with, and consumables and equipment can be easily found in normal biolabs. Here, the 3D imaging capabilities will be demonstrated only with commercial standard microscopes (e.g., spinning disk for 3D reconstruction and wide field microscopy for routine monitoring).

This paper presents a fabrication protocol for a new type of culture substrate with hundreds of microcontainers per mm2, in which organoids can be cultured and observed using high-resolution microscopy. The cell seeding and immunostaining protocols are also detailed.

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by ...

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

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