From stiff bone to soft fat and from miniscule capillaries to a whole brain, our cells’ ability to form myriad tissue types is the most fascinating yet challenging aspect of tissue engineering. In order to reproduce the complexity of the human body, tissue engineers will have to harness a variety of 3D bioprinting technologies instead of the current one-size-fits-all solutions. This post explains the advantages and disadvantages of two desktop three-dimensional (3D) bioprinting solutions offered by CELLINK—the extrusion-based bioprinters BIO X™ and INKREDIBLE™, and the light-based bioprinter Lumen X™, Powered by Volumetric—and specifically compares the mechanics, resolution, geometry and material compatibility of each.
Both bioprinting techniques begin with a computer-aided design (CAD) file that is sliced into discrete horizontal layers, which the printers then build and stack to produce the 3D construct. The difference lies in how each method treats those layers.
With extrusion-based bioprinting, the more common technique, a paste or fluid is loaded into a cartridge mounted on a gantry, or robotic arm, that moves along Cartesian coordinates above the printbed, or surface, the object will be printed on. A mechanical force, typically air pressure or a motor-driven piston, pushes the material through a nozzle to form a filament. By dragging the filament across the surface, the gantry traces the outline of the first layer. Then, as specified by the user, the gantry continues depositing filament layer by layer in an infill pattern to establish porosity and mechanical strength until the print is complete.
Digital light processing-based stereolithography (DLP-based SLA) is also a layer-by-layer process, but instead of extruding material through a nozzle, a source of illumination, not unlike a movie projector, treats each layer with a still image. Projecting this image into a bath or droplet of light-sensitive liquid stimulates a chemical reaction, which causes the liquid to solidify, or cure, only where illuminated. Stacking these cured layers on a build platform produces the printed object.
Often compared when discussing printing techniques is resolution, or the smallest theoretically printable detail, which is governed by a number of factors, such as material and geometry, that are outside the scope of this article. Our comparison focuses on planar resolution along the X and Y axes. In extrusion-based bioprinting, the diameter of the nozzle dictates the diameter of the filament that can be extruded. In DLP-based SLA, the size of the projected pixel defines the smallest point of light that can be cured. This point of light is typically smaller than most extrusion nozzle diameters and the chemical reaction is more consistent, allowing DLP-based SLA printing techniques to produce smaller and more intricate objects at a higher resolution than extrusion bioprinting. After the filament leaves the nozzle of an extrusion-based bioprinter, besides gravity and friction, nothing controls how the filament lays down and spreads, resulting in some variability along the boundary of the filament. Even with a filament that is the same size as the smallest point of light from a DLP-based projector, an extruded print will look rougher than an SLA-printed construct given this variable spread.
Since extruded prints are basically a stacking of cylinders, like cabin logs, the contact area between filaments is very small. When these stacked cylinders are themselves arranged in a tubular shape, like that of a blood vessel, a small contact area makes it difficult to ensure that the tube is watertight and strong enough to withstand the pressure of fluid flowing through it. DLP-based SLA, on the other hand, sandwiches sheets of printed materials in stacks, essentially glued from edge to edge, yielding significantly stronger, watertight structures. With stronger tubes and smoother sides, DLP-based SLA is well suited for bioprinting lab-on-a-chip microfluidic devices, especially ones with complicated networks or ones that need to be imaged, distortion-free, under a microscope. DLP-based SLA’s advantages when printing complex negative features like networks also contribute to its ability to print complex positive features like lattices.
Tissue engineering scaffolds need both a specific porosity and shape in all dimensions to match native tissue and allow cells to survive, but extrusion bioprinters create porosity with only the negative space between infill lines. When printing a cube, as shown in Figure 1, with an extrusion printer, a user might select a hexagonal infill of some percentage and the machine will print vertically aligned hexagons within the cube.
Figure 1. Extrusion 3D printing of a cube. A) CAD model of a cube, B) image of a slice showing perimeter (yellow) and infill (red) paths, C) the extruded cube.
While extrusion slicers focus on the outer bounds of an object, DLP-based SLA slicers capture the entire plane of a layer in a single image, operating with 100% infill, so that any porosity desired must be created in the original model. While this might pose upfront complications, many CAD suites can apply a lattice pattern to an object, such as the cube in Figure 2.
Figure 2. DLP-based 3D printing of a cube. A) CAD model of a cube with a gyroid structure, B) image of a slice that would be projected onto the photo-sensitive material, C) the printed gyroid cube (right).
Tissues, like bone, have porosities and geometries in three dimensions, so being able to generate and print a repeating lattice or randomized 3D structure will yield a scaffold that better mimics physiological tissue. The filament-by-filament extrusion-based approach has an inherent fragility that makes printing lattices, like the structure in Figure 2, nearly impossible. In addition, in extruded constructs, porosity exists vertically, while horizonal pores occur between layers and are therefore limited or nonexistent. Given the different strengths of each technique, users must consider the best suited method of bioprinting for their design. While extrusion offers a simplified design and print process that makes it well suited for labs with limited access to CAD software or just starting out, light-based bioprinting techniques are currently the best option for reproducing the smallest, most intricate structures of the human body. However, shape and size are only part of the tissue engineering puzzle.
Certainly, bioprinting organs would be far easier if organs were composed of one material and one cell type, but properly recreating an organ means capturing the spatial arrangement of different cell types. An extrusion printer can array any number of cartridges on a gantry, enabling the arrangement of materials and cells, filament by filament, that precisely mimics the in vivo environment. By contrast, DLP-based SLA baths are usually one material and one cell type. There have been multiple attempts to perform multi-material SLA printing in which the arrangement of the material or cell type does not matter; however, these attempts usually involve slow, repeated cleaning steps and run the risk of cross contamination between wash liquid or from one material to another. Extrusion allows for multi-material bioprinting in addition to making it possible to print with a wider variety of materials.
The simplicity of pushing fluids through a cartridge affords tissue engineers creativity and flexibility both before and after extrusion for liquefying then solidifying materials. The nearly infinite possibilities of extruded materials explains the variety of printheads available for the BIO X, allowing researchers to combine many materials and cell types in a single print. As the name implies, light-based bioprinting techniques, like DLP-based SLA found in the Lumen X, use photo-sensitive materials and a light source to promote solidification. The photo-sensitive material must also have a low enough viscosity to flow into a droplet as the build platform moves into and out of the liquid during the print.
When viewed through the lenses of mechanics, resolution, geometry and material selection, there are numerous differences between extrusion-based and DLP-based SLA bioprinters; however, the advantages of each make them perfectly complementary in many research environments. Extrusion offers multi-material printing from a vast palette of bioinks as well as simplified model designs, while DLP-based SLA is capable of remarkable geometric complexity at high resolution and, depending on the material used, perfect clarity. Researchers might also combine the two techniques in a single experiment. For example, by extruding a mix of cells from the BIO X into a microfluidic chip printed on the Lumen X. Whether you choose to add one or both to your workflow, you will find that CELLINK offers reliable, versatile bioprinting solutions.
Join us for a webinar on Tuesday, November 17th, with Professor Jordan Miller, Department of Bioengineering, Rice University, for an in-depth look at the Lumen X, Powered by Volumetric. Discover how the first DLP bioprinter can be used to develop multivascular networks and models that better represent life. Register here.