–Soft Is Hard
In the first part of this article series it was posited that although 3D bioprinting is approximately ten years old, it is already an area of major scientific hype despite its translation to the clinic remaining highly limited. The reason for this lack of translation was suggested to be technological barriers that must first be overcome before 3D bioprinting can begin executing on its lofty claims. By defining these barriers, we gated 3D bioprinting into four major stages, where overcoming one stage’s barrier allows the field to proceed to the next. To reiterate, the four stages are:
Stage 1: Lacks the ability to print using cells and/or proteins with high fidelity.
Stage 2: Prints cells and/or proteins at high fidelity, but no significant tissue function upon print completion.
Stage 3: Combines an immature 3D printed tissue with a coordinated maturation system, resulting in a functional tissue for use or study.
Stage 4: Produces a tissue or organ that is functional immediately upon print completion.
Part 1 of this series focused on Stage 1 bioprinting, where the field lacks the ability to precisely print the biomaterials we truly wish to use. To briefly summarize, these biomaterials, unlike the plastics and metals used in 3D printing, are too soft to physically support their weight in the air, resulting in significant print deformation and a loss of fidelity. To minimize this print deformation, researchers compromise on bioink formulations with a litany of additives to make materials more rigid at the cost of biological relevance, such as exposing methacrylate inks to UV light. This exposure might not cause the immediate decimation of all cells, but the risk of long-term DNA damage is now elevated while also producing covalent bonds that make cell-based matrix remodeling more difficult. Fortunately, recent advancements have made significant headway to overcoming these barriers and have resulted in a significant increase in bioprinting resolution and have initiated the transition from Stage 1 to Stage 2.
The two major methods of 3D bioprinting are extrusion-based and light-based printing, with each technique possessing unique advantages and disadvantages. It is possible to argue that some techniques are hybrids that first extrude a UV light-sensitive ink, but these techniques do not provide the astounding resolution possible with pure light-based techniques. Instead, they struggle with each technique’s disadvantage by suffering from the lower resolution of extrusion-based printing as well as the chemical limitations of light-based printing. Of the recent advancements, the most potentially impactful in extrusion-based and light-based bioprinting are Freeform Reversible Embedding of Suspended Hydrogels (FRESH)1,2, and Stereolithography Apparatus for Tissue Engineering (SLATE)3, respectively.
FRESH is an extrusion-based printing technique in which bioinks are extruded into a support bath instead of in open air(1,2). This support bath cushions the fluid ink as it is injected inside the bath by the printer, preventing it from collapsing (Fig. 1A). The bath has an important property in that it has yield stress – at rest, it behaves like a solid, but when enough force is exerted upon it, it starts to flow like a fluid. Common household products like mayonnaise, ketchup, and hair gel all have yield stresses. They do not flow unless you shake or squeeze their containers, overcoming their yield stress in the process. This yield stress means the FRESH support bath flows out of the way of the needle and ink that is being injected but returns to behaving like a solid shortly thereafter. This recovery prevents the ink from deforming under its own weight as is common in open-air printing techniques. Not only does the bath physically support the ink, but it also allows for chemical interactions between the bath and the ink. For example, fibrin is a key protein that comprises a major component of blood clots. For fibrin to be formed, its predecessor fibrinogen must be cleaved enzymatically by thrombin as part of the clotting cascade. Instead of mixing fibrinogen and thrombin together at once, fibrinogen can instead be injected into a bath containing trace amounts of thrombin to allow it to be 3D printed without deformation. The bath is made from gelatin which melts at body temperature, meaning upon print completion, the container is warmed, causing the support to melt away, gently releasing the print. This means that FRESH expands the list of chemistries that can be 3D printed. Inks that are sensitive to pH (collagens), ions (alginates), or enzymes (fibrinogen) can be printed into baths containing their respective pH buffer, ion, or enzyme that cause them to gel. By printing acidic collagen into a pH neutral bath, the pH-sensitive collagen gels, allowing researchers to bioprint early-stage heart valves, blood vessel networks, and more (2). Moreover, highly cell-dense constructs can be printed with FRESH whether the cells are in a compact ink (2) or bath (4) phase on the order of hundreds of millions of cells per milliliter.
SLATE is a light-based printing technique in which yellow food coloring (tartrazine) acts as a photo absorber while Lithium phenyl-2,4,6-trimethyl-benzoyl phosphinate (LAP) acts as a photoinitiator for a photo-sensitive polymer such as (poly(ethylene glycol) diacrylate [PEGDA])(3) (Fig. 1B). Long names aside, a critical aspect of light-based printing is that one needs a chemical that will start a polymer polymerizing (a photoinitiator) when exposed to UV light while also making sure that only the thickness you want to print solidifies, hence the use of a photo absorber to prevent stray light from scattering into the unpolymerized resin. Previous techniques used photo absorbers and photoinitiators that were more cytotoxic or had other compromises such as printing slower or less precisely. The optimizations in SLATE allow for quick printing at high resolution with less toxicity. With these improvements, researchers printed a model of a lung alveolus in which an air sac was surrounded by a complex 3D channel network to mimic the way capillaries surround alveoli to facilitate gas exchange.
This is not to say that either FRESH or SLATE is perfect. As economist Thomas Sowell put it, “There are no solutions, only trade-offs.”. The pros and cons to FRESH and SLATE are listed (Fig. 1 C and D). It is important to note that much of the pros and cons of these bioprinting techniques are now the same as the pros and cons of their regular plastic 3D printing counterpart techniques (FDM and SLA). This indicates that biological researchers have discovered the modifications they needed to make to traditional FDM and SLA techniques to translate 3D printing to 3D bioprinting.
Although bioprinting cells and proteins at high fidelity are starting to become possible, there is still a large disparity at the microscopic structural level between bioprinted and native tissues. Figure 2 seeks to compare notable bioprinted analogs of tissues to the actual microstructure of native tissue. The most crucial detail in this figure is to pay close attention to the scale bars when juxtaposing the native tissue and bioprinted microstructure. The level of microstructural detail of bioprinted tissues still falls well short of matching the microarchitecture of native tissue. The glomerulus of a kidney is still far more complex than a channel with kidney cells5 (Fig. 2 A to D). A dense capillary network wrapped around alveolar sacs is still orders of magnitude smaller than its SLATE-printed counterpart (Fig. 2 E to H). Cardiac muscle tissue contains muscle fibers, capillaries, and electrically conductive Purkinje fibers all organized in a dense fashion and it is still far smaller than what is printed with FRESH (Fig. 2 I to L). This lack of true microstructure is now the large barrier that prevents functional tissue from being 3D bioprinted. Now that we can 3D bioprint with high fidelity, the next challenge becomes printing functional tissue, which in turn means replicating tissue-specific microarchitecture. To expect 3D bioprinting to produce a functional, mature tissue immediately upon print completion is a rather unrealistic expectation. Every human is given 9 months of gestation to develop these organs that continue to mature for years after birth. Why should we expect engineered tissue to outperform developmental biology billions of years in the making? The next most reasonable step is to, therefore, combine an immature bioprinted tissue with a maturation system that seeks to encourage cells to assemble into the microarchitecture seen in native tissues, promoting function. Although a vast chasm of research must be crossed to get to Stage 3, that is where the translational potential explodes, and where 3D bioprinting must start to follow through on its promises.
1. Hinton, T. J. et al. Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels. Sci. Adv. 1, (2015).
2. Lee, A. et al. 3D bioprinting of collagen to rebuild components of the human heart. Science (80-. ). 365, 482–487 (2019).
3. Grigoryan, B. et al. Multivascular networks and functional intravascular topologies within biocompatible hydrogels. Science (80-. ). 364, 458–464 (2019).
4. Skylar-Scott, M. A. et al. Biomanufacturing of organ-specific tissues with high cellular density and embedded vascular channels. Sci. Adv. 5, (2019).
5. Homan, K. A. et al. Bioprinting of 3D Convoluted Renal Proximal Tubules on Perfusable Chips. Sci. Rep. 6, 1–13 (2016).
About the Author:
Chief Operations Officer, Fluidform
Andrew is a co-founder of FluidForm and leads the development, manufacturing, and scale-up efforts of LifeSupport™. His research focuses on developing the next generation of techniques for vascularizing 3D-bioprinted tissues to improve the clinical translational potential of tissue-engineered therapies.