Generation of Three-dimensional Printed Biological Innovative Mold for Optically Transparent Tissue Specimens

Summary

Tissue innovative Molds (iMolds) have been developed to reduce specimen movement, structurally support the specimen being imaged, and allow for repeated imaging on precise anatomical locations using optically-transparent samples.

Abstract

Histology continues to evolve in modern day experimentation. Recently, groups have been able to make tissue and bones transparent by removing lipids and other biological species that prevent deep tissue imaging. Now, groups can robustly "clear" the tissue or specimen and perform a wide array of microscopy such as single-photon, multiphoton, and light-sheet to evaluate their biological target of interest. This has eliminated the need to freeze, cyro-slice, and cyro-preserve tissue, greatly limiting human prone error. However, current tissue clearing methods still lack a robust system to mount and structurally support unique specimens. This leads to issues with tissue movement during imaging, the lack of reliability when re-staining and re-evaluating specimens, and working with abnormally shaped specimens such as tumors. To overcome these obstacles, the innovative Mold (iMold) was generated. iMolds are first created from images of the specimen followed by three-dimensional (3D) printed molds that support the unique tissue structure being imaged and re-imaged. This leads to a dramatic reduction in tissue movement while imaging and allows for scientists to robustly re-evaluate areas of interest that have been re-stained or need to be re-imaged. Furthermore, iMolds can be created in a short time period and generated for multiple samples in any imaging format (i.e. slide, cell culture plate). This allows users to scan multiple organs or specimens on one slide or cell culture dish. In closing, iMolds can be utilized by any research laboratory studying cleared specimens while also reducing costs, time, and tissue movement compared to other current methodologies.

Introduction

Whole tissue clearing methods have recently become a widely-used method in histology. First the tissue and organ are cleared resulting in the specimen becoming optically-transparent, then this is followed by staining and imaging. With these techniques scientists are able to image at high resolution entire organs and thick specimens. In addition, many tissue clearing methodologies allow for repeated staining. This provides users the ability to develop 3D renderings of structural and functional relationships across a large volume of area.

The 3D renderings that are produced have application in many fields of science. For example, scientists can now clear and generate a 3D model of a tumor at cellular and subcellular resolution¹. This allows scientists the ability to study the heterogeneity of cells, create 3D renderings of entire organs to examine cell interactions, vascularization, and environment niches. Furthermore, using systems like this also allows a whole organ evaluation after drug screenings and other therapeutic interventions. The benefits of tissue clearing make it imperative that there is a rapid and reliable method to structurally support any specimen being imaged to the micron level and also provide a method to place multiple organs on one imaging platform, to allow for screening of multiple specimens.

For these reasons, the tissue iMold has been generated². iMolds are made to be utilized by researchers evaluating large tissue specimens that are optically transparent. The iMold restricts all tissue movement during prolonged and repeated imaging. Current methodologies lead to specimen movement during imaging and re-imaging³. In addition, current methods make it near impossible for sequential staining of the same cellular environment, due to relocating precise anatomical locations. These caveats are resolved with the generation of the tissue specific iMolds.

iMolds can be generated by almost all 3D printers and used by any research laboratory. First, they are generated at the micron level with the material of choice, such as PLA plastic. Then the iMold is mounted and can be used for repeated imaging. iMolds also allow the user to image multiple organs on one imaging platform during one imaging session, or repeated sessions. They are inexpensive to generate and reliable to use. The development of the iMold and representative results are detailed in this protocol.

Protocol

1. Clear the Tissue or Bone Specimens Following Established Clearing Methods

1. Follow this protocol or another established tissue-clearing method: for passive CLARITY begin by making hydrogel monomer (HM) solution included of 1% (wt/vol) acrylamide, 0.0125 - 0.05% (wt/vol) bisacrylamide, 4% paraformaldehyde, 1x PBS, deionized water, and 0.25% of the thermal initiator into a 50-mL tube³.
2. Perform a trans-cardial perfusion with HM and incubate the tissue overnight or 2 days at 4 °C with the HM solution in a 50-mL tube³.
   NOTE: Add enough HM solution to completely incubate the tissue, typically 10 - 15 mL is sufficient.
3. De-gas the tissue in the 50-mL tube to replace the oxygen with nitrogen, followed by incubation at 37 °C for 3 - 4 h for hydrogel polymerization.
   NOTE: De-gas following the established protocol³. Remove the oxygen by placing an aspirator into the 50-mL tube. With the aspirator in place, put a line of nitrogen gas directly into the solution, causing it to bubble. Perform this action for 5 min followed by polymerization.
4. Clear the tissue in a 37 °C shaking incubator with a buffered clearing solution consisting of 4% (wt/vol) sodium dodecyl sulfate and 0.2 M boric acid (pH 8.5) with solution replacement every one to two days. After this step, wash off the clearing solution with a 0.2 M boric acid buffer (pH 8.5)/0.1% Triton X-100. Typically, 10 - 15 mL of solution is sufficient, ensure the tissue is completely submerged.
5. For storage, place specimens in 1x PBS or imaging solution until it is time to image.

2. Generate the Tissue iMold Image File

1. Place the specimen onto a flat surface (i.e. lab bench).
2. Measure the length, width, and thickness of the specimen.
   NOTE: Measuring the samples before clearing the specimen is preferred, as cleared tissue may be challenging to measure due to its transparency. However, measuring after clearing should also be performed, if the user notices tissue structure deformations post-tissue clearing.
3. Take a photo of the specimen with the measurements (e.g. cell phone camera).
4. Double-click to open a sterolithography (.STL) file creator.
5. Upload the current photo into the software by selecting "file" and select "open."
6. Now with the uploaded image, click and hold to select the image and drag the current image onto the current z-plane.
7. Click the image and scroll towards the image corner to shrink or outwards to maximize the image.
   NOTE: It is easier for measurements if the user makes the initial imported photo small and then proceed to adjust it after tracing.
8. On the toolbar select the "freehand" drawing tool.
9. With the freehand tool draw around the region or specimen of interest.
   NOTE: Write down the current dimensions to be used to speed up the process of making the current image.
10. Click the "pointer" button on the toolbar and select the image that was just traced.
11. Press "delete" on the keyboard and delete the original image (now there is a sketch of the specimen).
12. Select the "pointer" button on the toolbar and click the original image face.
13. Select the "scale" tool in the toolbar.
14. Select the specimen image face with the scale tool.Type in the proper width or length into the scale measurement box at the bottom right corner of the screen.
15. Repeat the prior step to complete both the width and the length of the specimen.
    NOTE: Now there will be an image face file of the exact same dimensions of the specimen being traced and used.
16. Click and select a region outside of the specimen face.
17. Click and select the "rectangle" tool.
18. With the rectangle tool draw a rectangle or square of the size of the slide or plate that is planned to be imaged on.
    NOTE: Slides are commonly used and are 25 mm × 75 mm. However, if the imaging platform is designed for cell culture plates, then design based on those measurements.
19. Select the "pointer" button on the toolbar and click and select the face of the specimen image.
20. Click and select the "move" tool from the toolbar.
21. Click the specimen and move the specimen face onto the face of the rectangle (same z-plane) by dragging the image.
    NOTE: Place the specimen in the innermost area of the iMold to allow for the gluing step to be performed without mixing of imaging medium and glue.
22. Select the "pointer" button on the toolbar and re-select the specimen face.
23. Press "delete" on the keyboard to remove the specimen face.
    NOTE: Now the rectangle will have an empty space for where the specimen will be placed.
24. In the toolbar select the "push/pull" tool.
25. Click and select the rectangle face with the specimen area previously deleted.
26. In the button left hand corner type in the measurement to be the thickness of the rectangle (i.e. the thickness of the tissue).
    NOTE: Now there will be a rectangle the size of a slide or cell culture plate to be imaged with a missing place for the specimen to be placed within. However, it is suggested to add 0.5 mm to each side of the specimen to allow for imaging media to be placed with the specimen, but this is not required.
27. In the "file" tab select the file and export it as a .STL.

3. Printing the .STL file iMold

1. Open the saved .STL file in the 3D printer software. Go to the "file" tab and select "open."
2. Select "print" from library on the button of the screen.
3. Select the .STL file.
4. Print the file at optimal resolution (i.e. 150 μM) with the highest quality setting.
   NOTE: It typically takes between 1 - 4 h depending on the specimen and imaging platform. PLA is commonly used for its flexibility but other plastics such as ABS also work for iMold generation. 3D printers with heated stages are preferred for improved printing results.

4. Using the .STL iMold

1. Place the printed iMold on the working lab bench.
2. Place a few lines of super glue around the specimen area.
   NOTE: Glue the outermost area of the iMold to avoid mixing of superglue with the specimen. In addition, using superglue that can be purchased in a pen style to allow a finer width to be applied.
3. Place coverglass onto the superglue.
   NOTE: Allow sufficient time for the glue to dry, if using an upright microscope then super glue must be applied to both sides with coverglass once the specimen and liquid has been placed in the iMold.
4. Place the specimen into the iMold.
5. Add the appropriate amount of liquid of clearing solution to fill the chamber of the iMold but do not add excess as this will mix with the superglue.
   NOTE: The clearing solution refractive index should match the refractive index of the objective being used to image for optimal imaging results.
6. Proceed to imaging (step 5).
   NOTE: If re-staining, remove the specimen, clear the prior antibodies following the previously established protocols, re-stain, and replace the specimen into the iMold and re-image. If the iMold becomes structurally altered, then re-print a new iMold from the saved .STL file.

5. Imaging the iMold

1. Place the tissue iMold into the appropriate microscope (i.e. single-photon confocal or multi-photon) platform (i.e. plate holder, slide holder). Add imaging solution until the entire iMold chamber is filled, typically less than 200 µL is needed.
2. Find the location of interest and image using the ideal microscope settings.
   NOTE: Imaging using the z-stack tile allows for a large area to be imaged through multiple z-planes. In addition, ensure proper controls when using new antibodies.

Results

The benefits of this method, in combination with tissue clearing methods is the ability to control movement of tissue to avoid shifting during long term imaging and/or the re-staining of tissue. Therefore, it is critical that the user designs a properly fitted iMold, have proper tissue-clearing, and have the validation of the immunofluorescent target (i.e. RNA, Protein). Other methods include using putty or iSpacers to maintain the tissue, however this leads to the issues previou...

Discussion

Optical clearing methods are continuing to increase the understanding of environmental niches and cellular heterogeneity. The tissue is maintained and stored in a hydrogel complex that allows biomacromolecules to permeate the matrix. This allows for groups to study immunofluorescence of proteins and nucleic acids either by endogenous fluorescence or post-hoc labeling, across large volumes of tissue.

However, a major caveat in the system comes when attempting to image the tissue. In current met...

Materials

Name	Company	Catalog Number	Comments
Acrylamide	Sigma	A9099
bisacrylamide	VWR	0172-50G
paraformaldehyde	Electron Microscopy Sciences	15714-S
PBS	Quality Biological	119-069-131
thermal initiator	Wako Chemicals	VA-044
sodium dodecyl sulfate	Bio-Rad	161-0302
boric acid	Sigma	B-7660
Triton X-100	Sigma	X100
focus clear	CEDARLANE	F101-KIT
Google Sketchup
Camera			we used an iPhone 7 camera
3D Printer	M3D	Micro +
PLA plastic roll for 3D Printer	M3D	3D Ink	no catalog number listed, we use black PLA filament