Preparation & Staining of 3D Printed Tissues & Scaffolds

Introduction

Three-dimensional (3D) printing of biological tissue is rapidly becoming an integral part of tissue engineering. With advances in 3D printing technology, numerous novel fabrication methods are now possible, allowing for the production of cell laden constructs,1-2 composite tissue2 as well as scaffold-free tissue3. These advances bring us one step closer to printing whole functional organs from raw materials. 3D printing can also be used to create tissue engineering scaffolds that mimic the 3D microenvironment of the native organ. For fabrication of tissue scaffolds, 3D printing is amenable to a wide range of biomaterials such as calcium phosphate, 4 CaSiO3,5 collagen,4 hydrogel,6 hydroxyapatite,7-8 polycaprolactone (PCL),2,8 as well as starch-based polymers.9 This flexibility allows fabrication of composite structures that best mimic the physiological conditions surrounding the tissue, including topographical cues as well as mechanical properties.10 Thanks to these properties, 3D printed scaffolds serve as desirable support structures for 3D cultivation of cells.

On the other hand, the characterization of 3D printed tissue often involves preparatory steps that are not used in conventional 2D tissue culture analysis. For example, imaging through a thick 3D tissue using light microscopy techniques, such as confocal microscopy, is challenging due to scattered light in biological samples. Because of this, many imaging approaches are applied to tissue sections that are less than 20 µm thick.11 While innovative imaging approaches report successful visualization of much thicker tissues, they are still limited to tissue thickness of several millimeters at most.11 Therefore, proper sectioning is important for characterization of multilayer cell constructs produced by 3D printing. Biological stains should be carefully selected in order to extract the most relevant information about the printed tissue, such as cell viability and maturation. For instance, determination of cell viability in the inner core of a 3D printed construct is important as these cells may have a greater chance of necrosis due to insufficient nutrient exchange.12 In this technology spotlight, we briefly discuss processing and staining approaches for evaluation of 3D printed tissues and scaffolds.

Confirming material compatibility with cells of interest

Before optimizing a 3D printing technique for a specific biological application, it may be beneficial to confirm the material compatibility with the cells of interest to ensure that cells attach and proliferate on the printed material. To do so, cell viability and proliferation assays can be performed on cells cultured on the raw material. For more information on cell viability and proliferation assays, follow this link.

Preparation of 3D printed tissue/scaffold for light microscopy

Once a cellular construct has been successfully printed or grown on a scaffold, a chemical fixative (Table 1) is applied prior to embedding and sectioning to prevent change or loss of cellular components during processing. Alternately, tissue may be frozen and cryo-sectioned if chemical fixation is not desired. While the use of formalin solution is popular in tissue fixation,13 the choice of fixative depends highly on the specific properties of the tissue and the purpose of the study. Factors that may affect fixation include the mechanism of the fixative (e.g. crosslinking, denaturation, etc) 13 as well as the condition of the fixation procedure (e.g. temperature and duration of tissue exposure to fixative).13-14 Non-toxic agents that do not contain formaldehyde or glutaraldehyde are also available for tissue fixation (HistoChoice® tissue fixative (Product No. H2904) and clearing agent (Product No. H2779)). A complete list of tissue processing products can be found here.

Table 1. Various fixatives used for tissue fixation
 


Once properly fixed, the tissue can be sequentially dehydrated in ethanol (Product No. E7023) and embedded in a medium such as Paraplast® (Table 2) for sectioning.13 The embedded tissue can be sectioned in a desired thickness onto glass slides (Table 2) using a microtome. Sectioning thickness should be determined based on the specific type of the printed tissue as well as the biological stains that will be used. For example, while muscle tissues can be sectioned in 4-6 µm slices, brain or spinal cord samples are recommended to be cut in thicker (10-40 µm) slices.15

Table 2. Materials used for processing and embedding tissue samples
 

Product No. Description Purpose
P3558 Paraplast® Tissue embedding (cuts to 4 μm thickness)
P3808 Paraplast X-TRA® Tissue embedding (cuts to 2 μm thickness)
P3683 Paraplast Plus® Tissue embedding for tissues that are difficult to process with Paraplast®
E7023 Ethanol, pure Dehydration
534056 Xylene (histological grade) Removal of paraffin
S8902 (plain)
S8400 (frosted)
S9027 (opaque)
Glass slides Microscope slides for tissue handling
C9802 (22x22mm)
C7931 (24x40mm)
C8181 (24x50mm)
C9056 (24x60mm)
Cover glasses Cover glass for microscope slides
P5493 Phosphate buffered saline (PBS) Common washing buffer


Once the tissue is sectioned onto microscope slides, various staining procedures can be performed using stains and buffers listed in Table 3. For a specific antigen of interest, immunohistochemistry (IHC) techniques may be utilized. For more information on IHC, click on this link. Following IHC, samples are often co-stained with a counterstain, such as DAPI (Product No. D9542) or hematoxylin (Product No. HHS16).

Table 3. Popular biological stains and buffers
 

Product No. Description Purpose
D9542
DAPI
Nucleic acid staining
P5282 (FITC)
P1951 (TRITC)
Fluorophore conjugated phalloidin Stains F-actin of the cell cytoskeleton
T6146 (powder)
T8154 (soln)
Trypan blue Used in cytotoxicity and proliferation assays
81845 (powder)
P4864 (soln)
Propidium iodide (PI) Counterstaining
HHS16 (500mL)
HHS32 (1L)
HHS80 (2.5L)
HHS128 (4L)
Hematoxylin solution, Harris , modified
Counterstaining
P5493
Phosphate buffered saline (PBS)
Common washing buffer
A9647
Bovine serum albumin (BSA)
Component of blocking buffer
P3688
PBS with 1% BSA
Blocking buffer/diluent
P9416
TWEEN® 20
Component of blocking buffer/diluent
T8787 Triton X™ Component of permeabilization buffer

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

In addition to light microscopy techniques described above, SEM analysis may be necessary to visualize nanoscale morphology of the 3D printed construct. SEM imaging is particularly useful for 3D printed scaffolds that are designed to mimic the micro/nanostructures of the tissue microenvironment. Preparation of biological samples for SEM involves fixation in EM grade fixatives (Table 4), sequential dehydration in ethanol (Product No. E7023), critical point drying, and coating with a conductive material (e.g. gold) to reduce charging artifacts.

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

Product No. Description
G7776 (70%)
G7651 (50%)
G5882 (25%)
G7526 (8%)
Glutaraldehyde solution (Grade I, purified for use as an EM fixative)
E7023 Ethanol, pure

 

References

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Advanced Materials (FRG) 2014-05-21
A new bioprinting method is reported for fabricating 3D tissue constructs replete with vasculature, multiple types of cells, and extracellular matrix. These intricate, heterogeneous structures are created by precisely co-printing multiple materials, known as bioinks, in three dimensions. These 3D micro-engineered environments op...Read More
Jung-Seob Lee, Jung Min Hong, Jin Woo Jung, Jin-Hyung Shim, Jeong-Hoon Oh, Dong-Woo Cho
Biofabrication 2014-06-01
In the ear reconstruction field, tissue engineering enabling the regeneration of the ear's own tissue has been considered to be a promising technology. However, the ear is known to be difficult to regenerate using traditional methods due to its complex shape and composition. In this study, we used three-dimensional (3D) printing...Read More
Cyrille Norotte, Francois S Marga, Laura E Niklason, Gabor Forgacs
Biomaterials 2009-10-01
Current limitations of exogenous scaffolds or extracellular matrix based materials have underlined the need for alternative tissue-engineering solutions. Scaffolds may elicit adverse host responses and interfere with direct cell-cell interaction, as well as assembly and alignment of cell-produced ECM. Thus, fabrication technique...Read More
Jason A Inzana, Diana Olvera, Seth M Fuller, James P Kelly, Olivia A Graeve, Edward M Schwarz, Stephen L Kates, Hani A Awad
Biomaterials 2014-04-01
Low temperature 3D printing of calcium phosphate scaffolds holds great promise for fabricating synthetic bone graft substitutes with enhanced performance over traditional techniques. Many design parameters, such as the binder solution properties, have yet to be optimized to ensure maximal biocompatibility and osteoconductivity w...Read More
Chengtie Wu, Wei Fan, Yinghong Zhou, Yongxiang Luo, Michael Gelinsky, Jiang Changa, Yin Xiao
Journal of Materials Chemistry 2012-05-01
Calcium silicate (CaSiO3, CS) ceramics have received significant attention for application in bone regeneration due to their excellent in vitro apatite-mineralization ability; however, how to prepare porous CS scaffolds with a controllable pore structure for bone tissue engineering still remains a challenge. Conventional methods...Read More
L A Hockaday, K H Kang, N W Colangelo, P Y C Cheung, B Duan, E Malone, J Wu, L N Girardi, L J Bonassar, H Lipson, C C Chu, J T Butcher
Biofabrication 2012-09-01
The aortic valve exhibits complex three-dimensional (3D) anatomy and heterogeneity essential for the long-term efficient biomechanical function. These are, however, challenging to mimic in de novo engineered living tissue valve strategies. We present a novel simultaneous 3D printing/photocrosslinking technique for rapidly engine...Read More
Barbara Leukers, Hülya Gülkan, Stephan H Irsen, Stefan Milz, Carsten Tille, Matthias Schieker, Hermann Seitz
Journal of Materials Science: Materials in Medicine 2005-12-01
Nowadays, there is a significant need for synthetic bone replacement materials used in bone tissue engineering (BTE). Rapid prototyping and especially 3D printing is a suitable technique to create custom implants based on medical data sets. 3D printing allows to fabricate scaffolds based on Hydroxyapatite with complex internal s...Read More
Su A Park, Su Hee Lee, Wan Doo Kim
Bioprocess and Biosystems Engineering 2011-05-01
For tissue engineering and regeneration, a porous scaffold with interconnected networks is needed to guide cell attachment and growth/ingrowth in three-dimensional (3D) structure. Using a rapid prototyping (RP) technique, we designed and fabricated 3D plotting system and three types of scaffolds: those from polycaprolactone (PCL...Read More
C.X.F Lam, X.M Mo, S.H Teoh, D.W Hutmacher
Materials Science and Engineering: C 2002-01-01
Rapid prototyping (RP) techniques have been utilised by tissue engineers to produce three-dimensional (3D) porous scaffolds. RP technologies allow the design and fabrication of complex scaffold geometries with a fully interconnected pore network. Three-dimensional printing (3DP) technique was used to fabricate scaffolds with a...Read More
Sean V Murphy, Anthony Atala
Nature Biotechnology 2014-08-01
Additive manufacturing, otherwise known as three-dimensional (3D) printing, is driving major innovations in many areas, such as engineering, manufacturing, art, education and medicine. Recent advances have enabled 3D printing of biocompatible materials, cells and supporting components into complex 3D functional living tissues. 3...Read More
Benjamin Gantenbein-Ritter, Christoph M Sprecher, Samantha Chan, Svenja Illien-Jünger, Sibylle Grad
Methods in Molecular Biology 2011-01-01
In tissue engineering, a variety of methods are commonly used to evaluate survival of cells inside tissues or three-dimensional (3D) carriers. Among these methods confocal laser scanning microscopy opened accessibility of 3D tissue using live cell imaging into the tissue or 3D scaffolds. However, although this technique is ideal...Read More
Craig K Griffith, Cheryl Miller, Richard C A Sainson, Jay W Calvert, Noo Li Jeon, Christopher C W Hughes, Steven C George
Tissue Engineering (United States) 2005-01-01
Although tissue engineering promises to replace or restore lost function to nearly every tissue in the body, successful applications are currently limited to tissue less than 2 mm in thickness. in vivo capillary networks deliver oxygen and nutrients to thicker (> 2 mm) tissues, suggesting that introduction of a preformed in vitr...Read More
William J Howat, Beverley A Wilson
Methods 2014-11-01
It is impossible to underplay the importance of fixation in histopathology. Whether the scientist is interested in the extraction of information on lipids, proteins, RNA or DNA, fixation is critical to this extraction. This review aims to give a brief overview of the current "state of play" in fixation and focus on the effect fi...Read More
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North American journal of medical sciences 2010-05-01
This article summarized immunohistochemistry methods generally used in research laboratories and clinic including direct immune staining, indirect immune staining, enzyme method, fluorescence method, APC method and PAP method.Read More