How are 3D printed tissues & scaffolds prepared and stained?

The three-dimensional (3D) printing of biological tissue is quickly becoming a vital part of tissue engineering. Advancements in 3D printing technology have facilitated the development of many new fabrication methods, enabling the production of cell-laden constructs,1-2 composite tissue,2 and scaffold-free tissue.3 These advances pave the way for the eventual printing of whole functional organs from raw materials.

Image Credit: asharkyu/Shutterstock.com

Additionally, 3D printing can be employed to produce tissue engineering scaffolds that replicate the 3D microenvironment of the native organ. For the fabrication of tissue scaffolds, 3D printing is amenable to a variety of biomaterials, including collagen, calcium phosphate,4 hydrogel,6 polycaprolactone (PCL),2,8 CaSiO3,5 hydroxyapatite,7-8 and starch-based polymers.9

This flexibility enables the fabrication of composite structures that best replicate the physiological conditions that surround the tissue, such as mechanical properties and topographical cues.10 Due to these properties, 3D-printed scaffolds can act as desirable support structures for the 3D cultivation of cells.

However, characterizing 3D printed tissue frequently requires preparatory steps that are not employed in conventional 2D tissue culture analysis. For instance, employing light microscopy methods like confocal microscopy to image through thick 3D tissue is difficult because of scattered light in biological samples. As a result, many imaging techniques are applied to tissue sections that have a thickness of less than 20 µm.11

Although innovative imaging methods report successful visualization of much thicker tissues, they are still limited to tissues that are at most several millimeters thick.11 Therefore, proper sectioning is important for the characterization of multilayer cell constructs produced by 3D printing.

Biological stains should be carefully chosen to extract the most relevant information about the printed tissue, including cell viability and maturation. For example, determining the cell viability in the inner core of a 3D printed construct is crucial as these cells can have a higher likelihood of necrosis caused by insufficient nutrient exchange.12

This article discusses the processing and staining approaches for the evaluation of 3D-printed tissues and scaffolds.

Confirming material compatibility with cells of interest

Before fine-tuning a 3D printing method for biological use, it is important to check that the material works well with the specific cells, allowing them to stick to it and grow. This can be done by testing cell survival and growth on the material before printing.

Preparing 3D printed tissues/scaffold for light microscopy

Once a cellular construct has been successfully printed or grown on a scaffold, a chemical fixative (detailed in Table 1) is applied before any embedding or sectioning takes place. This is to prevent any change or loss of cellular components during processing.

Alternatively, tissue can be frozen and cryo-sectioned if chemical fixation is not desired.

Although the utilization of formalin solution is common in tissue fixation,13 the choice of fixative is highly dependent on the tissue’s specific properties as well as the purpose of the study.

Factors that can influence fixation include the mechanism of the fixative (e.g., denaturation or crosslinking) 13 and the condition of the fixation procedure (e.g., duration of tissue exposure to fixative and temperature).13-14 

Additionally, non-toxic agents that do not contain glutaraldehyde or formaldehyde are available for tissue fixation, such as the HistoChoice® tissue fixative and clearing agent from Merck. Table 1 details available tissue processing products.

Table 1. Various fixatives used for tissue fixation. Source: Merck 

Product No. Description
340855 Glutaraldehyde solution 50 wt. % in H2O
HT501128 Formalin solution, neutral buffered, 10% histological tissue fixative
P6148 Paraformaldehyde reagent grade, crystalline
H0290 Hartman′s Fixative histological tissue fixative
Z2902 Zinc Formalin Fixative
A5472 Formalin Free Tissue Fixative  
2858 Ethanol Fixative 80% v/v suitable for fixing solution (blood films)
H2904 HistoChoice® Tissue Fixative
H2779 HistoChoice® Clearing Agent

 

After being properly fixed, the tissue can be sequentially dehydrated in ethanol and embedded in a medium like Paraplast® (detailed in Table 2) for sectioning.13 The embedded tissue may be sectioned in a desired thickness onto glass slides (as shown in Table 2) via a microtome.

The thickness of sections must be determined based on the specific type of printed tissue and the biological stains that will be utilized. For instance, while muscle tissues can be sectioned in slices that are 4-6 µm in thickness, it is recommended that brain or spinal cord samples are sectioned into thicker (10-40 µm) slices.15

Table 2. Materials used for processing and embedding tissue samples. Source: Merck 

Product No. Description
P3558 Paraplast® for tissue embedding
P3808 Paraplast X-TRA® for tissue embedding
P3683 Paraplast Plus® for tissue embedding
E7023 Ethyl alcohol, Pure 200 proof, for molecular biology  
534056 Xylenes histological grade
S8902 Slides, microscope plain, size 25 mm × 75 mm
S8400 Slides, microscope frosted one end, size 25 mm × 75 mm
S9027 Slides, microscope opaque (white), size 25 mm × 75 mm
C9802 Cover glasses size 22 mm × 22 mm
C7931 Cover glasses size 24 mm × 40 mm
C8181 Cover glasses size 24 mm × 50 mm
C9056 Cover glasses size 24 mm × 60 mm
P5493 Phosphate buffered saline 10× concentrate, BioPerformance Certified, suitable for cell culture

 

Once the tissue is sectioned onto microscope slides, different staining procedures may be applied using the buffers and stains detailed in Table 3. For a specific antigen of interest, immunohistochemistry (IHC) methods may be employed.

Following IHC, samples are frequently co-stained using a counterstain, such as hematoxylin or DAPI.

Table 3. Popular biological stains and buffers. Source: Merck 

Product No. Description
D9542 DAPI for nucleic acid staining
P5282 Phalloidin, Fluorescein Isothiocyanate Labeled sequence Amanita phalloides(synthetic: peptide sequence)
P1951 Phalloidin–Tetramethylrhodamine B isothiocyanate sequence from Amanita phalloides(synthetic: peptide sequence)
T6146 Trypan Blue powder, BioReagent, suitable for cell culture
T8154 Trypan Blue solution 0.4%, liquid, sterile-filtered, suitable for cell culture
81845 Propidium iodide ≥94% (HPLC)
P4864 Propidium iodide solution solution (1.0 mg/ml in water)
HHS16 Harris Hematoxylin Solution, Modified
HHS32 Harris Hematoxylin Solution, Modified
HHS80 Harris Hematoxylin Solution, Modified
HHS128 Harris Hematoxylin Solution, Modified
P5493 Phosphate buffered saline 10× concentrate, BioPerformance Certified, suitable for cell culture
A9647 Bovine Serum Albumin heat shock fraction, pH 7, ≥98%
P3688 Phosphate buffered saline pH 7.4, contains BSA, powder
P9416 TWEEN® 20 for molecular biology, viscous liquid
T8787 Triton X-100 for molecular biology

 

Imaging 3D printed tissue/scaffold using scanning electron microscopy (SEM)

In addition to the above-mentioned light microscopy methods, SEM analysis may be required for the visualization of the 3D printed construct’s nanoscale morphology. SEM imaging is beneficial for 3D-printed scaffolds that are created to replicate the micro/nanostructures of the tissue microenvironment.

For SEM preparation, biological samples undergo a series of steps. First, they are fixed using EM grade fixatives (refer to Table 4), followed by sequential dehydration with ethanol. Next, they are subjected to critical point drying. Finally, the samples are coated with a conductive material, such as gold, to minimize charging artifacts.

Table 4. Chemicals used for fixation and dehydration for SEM imaging preparation. Source: Merck 

Product No. Description
G7776 Glutaraldehyde solution Grade I, 70% in H2O, specially purified for use as an electron microscopy fixative or other sophisticated use
G7651 Glutaraldehyde solution Grade I, 50% in H2O, specially purified for use as an electron microscopy fixative or other sophisticated use
G5882 Glutaraldehyde solution Grade I, 25% in H2O, specially purified for use as an electron microscopy fixative
G7526 Glutaraldehyde solution Grade I, 8% in H2O, specially purified for use as an electron microscopy fixative or other sophisticated use
E7023 Ethyl alcohol, Pure 200 proof, for molecular biology

 

References

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  2. Lee J, Hong JM, Jung JW, Shim J, Oh J, Cho D. 3D printing of composite tissue with complex shape applied to ear regeneration. Biofabrication. 6(2):024103. https://doi.org/10.1088/1758-5082/6/2/024103
  3. Norotte C, Marga FS, Niklason LE, Forgacs G. 2009. Scaffold-free vascular tissue engineering using bioprinting. Biomaterials. 30(30):5910-5917. https://doi.org/10.1016/j.biomaterials.2009.06.034
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  5. Wu C, Fan W, Zhou Y, Luo Y, Gelinsky M, Chang J, Xiao Y. 2012. 3D-printing of highly uniform CaSiO3 ceramic scaffolds: preparation, characterization and in vivo osteogenesis. J. Mater. Chem.. 22(24):12288. https://doi.org/10.1039/c2jm30566f
  6. Hockaday LA, Kang KH, Colangelo NW, Cheung PYC, Duan B, Malone E, Wu J, Girardi LN, Bonassar LJ, Lipson H, et al. 2012. Rapid 3D printing of anatomically accurate and mechanically heterogeneous aortic valve hydrogel scaffolds. Biofabrication. 4(3):035005. https://doi.org/10.1088/1758-5082/4/3/035005
  7. Leukers B, Gülkan H, Irsen SH, Milz S, Tille C, Schieker M, Seitz H. 2005. Hydroxyapatite scaffolds for bone tissue engineering made by 3D printing. J Mater Sci: Mater Med. 16(12):1121-1124. https://doi.org/10.1007/s10856-005-4716-5
  8. Park SA, Lee SH, Kim WD. 2011. Fabrication of porous polycaprolactone/hydroxyapatite (PCL/HA) blend scaffolds using a 3D plotting system for bone tissue engineering. Bioprocess Biosyst Eng. 34(4):505-513. https://doi.org/10.1007/s00449-010-0499-2
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  10. Murphy SV, Atala A. 2014. 3D bioprinting of tissues and organs. Nat Biotechnol. 32(8):773-785. https://doi.org/10.1038/nbt.2958
  11. Gantenbein-Ritter B, Sprecher CM, Chan S, Illien-Jünger S, Grad S. 2011. Confocal Imaging Protocols for Live/Dead Staining in Three-Dimensional Carriers.127-140. https://doi.org/10.1007/978-1-61779-108-6_14
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Last updated: Jan 15, 2024 at 5:48 AM

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