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

Summary

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.

Abstract

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.

Introduction

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 scarring^2,3. 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 healing^4,5,6. 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 gel^7,8,9. 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.

Protocol

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

1. Design the 3D printed holders and cover bar accounting for the average diameter of mouse globes and 3D print the design (Figure 1A, B).
2. 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.
3. Size the holder cover bar to the length of the holder'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.
4. Print the holder and cover bar.

2. Sample collection

1. 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.
2. 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.
3. 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.
4. 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 °C, 5% CO₂ incubator with low light conditions. Ensure the globes are submerged in the staining medium for uniform staining.
   1. 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 µg/mL bovine pituitary extract, 0.02 nM epidermal growth factor, 0.3 mM CaCl₂, and penicillin-streptomycin (100 units/mL and 100 µg/mL, respectively).
      ​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.

3. Preparation of sample holders

1. 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.
2. 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.
3. 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.

4. Wounding of the eye globes

1. 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.
2. Wound the globes using a sterile 25 G needle in the region of interest.
   1. 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.
   2. 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.
      ​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.

5. Sample placement on the holder

1. 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).
2. 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.
3. Adhere the 3D printed cover to the holder using glue, ensuring stabilization. Ensure the cover bar adheres to the holder and not the globe.
   ​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.

6. Sample imaging

1. Turn on the microscope and environmental chamber and verify that the chamber is humidified. Set the environmental chamber to 35 °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.
2. Place the coverslip, holder, and stabilized globe on the microscope stage within the environmental chamber and image using live cell imaging techniques⁹.
3. 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.
4. 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.
5. Record and save data in the preferred file format. The software used by the microscope produces .czi files for data recording.
6. Dispose of the globes as per the standard institutional protocols at the end of the protocol.

Results

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...

Discussion

This protocol describes a 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...

Materials

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