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.

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.

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

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