A Fresh Overview of FRESH Bioprinting
We see in sci-fi movies people living with organs and prosthetics crafted in sophisticated cell-deposition machines in a matter of seconds. But is our current technology even close to reaching this state?
In this article, I will be telling you everything you need to know about one of the most advanced currently bioprinting techniques called FRESH that is bound to change the world of healthcare and medicine. This will be a comprehensive article, so hold on tight!😉
What is 3D printing?
3D printing is fabricating 3D models based on a digital image of the structure using a machine called 3D printer.
Many types of 3D printing technology exist, but the most common 3D printers use a technique called Fused Deposition Modeling (FDM), where they extrude solid materials like plastic by melting them at the nozzle and depositing it on a flat surface layer-by-layer.
Depending on the model being printed, there may be overhangs that protrude out from the main body of the model. Just like how bridges can’t just hang in the air without any legs to support their weight, overhangs need support structures to uphold it while the molten plastic hardens. The need for overhangs requires additional labor after printing to remove the support structure, not to mention the vast amount of printing material that would be wasted in this process.
What is bioprinting?
Bioprinting is a subarea of 3D printing with the goal of printing cells and biomaterials in 3D space to recreate the structure and function of biological structures like organs. Whereas “conventional” 3D printing techniques create models by melting solid materials, bioprinters print out special materials called bioinks. Before further explaining what bioinks are, here’s a quick rundown of two essential components of bioinks for fully understanding them:
What are hydrogels?
You may have heard of (and maybe have eaten) gelatin 🍮 and alginate 🌿. These are both types of a material called hydrogel — a 3D net of hydrophilic (electrically charged) polymers that swell by trapping water molecules within their nets.
Hydrogels undergo a curing process called gelation that turns the individual polymer molecules into one interconnected net. During gelation, the polymeric material (pre-hydrogel substance composed of polymers) forms chemical bonds between individual polymer molecules in a process called crosslinking and traps water molecules on all four sides. The speed of gelation determines how quickly a layer of extruded bioink cures before the next layer is deposited onto it, and thus whether the print material would cure in the shape that it was extruded in.
What are cells?
Our body consists of hundreds of trillions of cells. Cells are the smallest units of life that grow, consume nutrients, exchange essential molecules and reproduce in a highly controlled way. All operations in a cell are coded into the cell using a programming language called — you guessed it — DNA, and cells communicate with one another by sending chemical messengers through an intercellular medium called the Extracellular Matrix (ECM). The ECM glues similarly functioning cells together into layers of cells called tissues, and tissues of related characteristics are associated into organs. Essentially, ECM is crucial for cell survival and the formation of organs. (Surprise, ECM’s also a type of hydrogel made of proteins called collagen).
Now back to bioprinting. Bioinks are mixtures of cells, the ECM, and organic hydrogel. “Okay, I get how the organs are made out of cells and the ECM helps keep the cells alive, but why do we need organic hydrogel?” Well, the hydrogel serves three main purposes: as filler material for empty spaces, medium to store nutrients to feed the cells (thanks to their similar material properties as the ECM), and a 3D structure to physically support the cells. The bioink could also include crosslinkers (molecules that become the “bridge” between polymer molecules) in the bioink to facilitate the gelation process.
Since we’re printing from containers of liquid bioink instead of plastic filament, we need a different way to extrude it (you definitely wouldn’t want to cook up the cells at the hot nozzle tip 😱!). Bioprinters, therefore, use the syringe to fine-tune the amount of hydrogel pushed out of the nozzle at a given time. The extruder controls the speed and degree to which the plunger is pushed into the barrel (see image below) to adjust the thickness of the deposited bioink, also called the layer height.
Here’s another problem that we need to solve: bioinks are soft (and not solid)! In other words, it’s impossible to construct a 3D structure by squeezing out 💉 the bioink in the air onto a petri dish. The extruded bioink structure would collapse before it can cure and the structure would be physically unstable throughout the printing process. Previously, scientists tried thickening the bioink with additives, but it resulted in lower print quality (thick bioinks could cause clogging in the nozzle) and ruined the structure’s biological properties of the result. Here is when the hero comes into the scene: FRESH bioprinting 😎
What is FRESH?
FRESH, short for Freeform Reversible Embedding of Suspended Hydrogels, refers to extruding hydrogel into a support bath that locks the hydrogel-based bioink (the “Hydrogel” part of the name) in place until it cures. In general, 3D printing techniques like FRESH where you print into a support bath is called embedded 3D printing. Remember the support structure used in fused deposition modeling? The support bath is like the support structure but one acting upon all surfaces of the extruded bioink and without any bioink wasted as support.
Support bath
The support bath exhibits a Bingham Plastic behavior (also called yield-stress behavior) which describes certain materials that act as a solid until they experience a shear force above a certain threshold that causes them to transition to a liquid-like behavior. (Fun fact: examples of Bingham plastic include mayonnaise and toothpaste.) The Bingham plastic behavior makes the support bath perfect for their job, which is maintaining the position of the printed bioink as they are extruded while letting the printer nozzle freely traverse through the bath.
Okay cool, but what is the support bath made of? Hydrogel microparticles, most commonly gelatin microparticles of ~60 micrometers in diameter, and a surrounding liquid phase (also called aqueous phase). To give you some context for what micrometer-scale particles feel like, the support bath has a similar texture as hair gel 👱🏼.
The liquid phase is a solution that the microparticles are submerged in. If you’ve tried Kona Ice before, you can think of the liquid phase as a syrup that you put on the ground ice (gelatin microparticles). The Kona Ice turns blue/red/whatever because the syrup seeps into the tiny gaps between the ice particles. Like the food coloring of the syrup, you can add materials to the aqueous phase to control the gelation mechanism of the bioink or provide nutrients to the cells. Although I mentioned earlier that cross-linkers are sometimes added to the bioink, they can also be added to the support bath. The latter is preferred since it allows for rapid crosslinking as soon as the bioink is exposed to the liquid phase and a long lifespan of the bioink since the ink would cure only after it is extruded (and not inside the syringe barrel).
The main advantage of using gelatin microparticles over other hydrogel particles is its stability at room temperature and melting temperature at 37 degrees Celsius. When the printing job is completed, the print chamber is heated to 37 degrees Celsius (a cell-friendly temperature), and the molten support bath is gently washed away. Don’t worry, there are no support structures to cut off 😉.
To recap, FRESH 3D bioprinting is depositing soft bioinks layer-by-layer into a chamber filled with support material that provides mechanical support on all sides of the printed filament.
Advantages of FRESH
What makes FRESH bioprinting so AMAZING is the possible resolution, size, geometric complexity, and range of printable materials achieved — aspects that take us a step closer to the ultimate goal of 3D printing organs ❤️ 👂💪.
Previously, only bioinks of certain rheological characteristics (ex. viscosity) could be printed due to the effects of gravity on print quality. However, implementing the support bath technology that eliminates this force of gravity on the printed material allows us to print with rheologically unmodified bioinks, and in turn, engineer functioning tissues. In addition, the protection from gravity enables the printing of larger soft structures, which break easily without any physical support.
Since the support bath holds up the printed structure in all three dimensions, the print nozzle can now move simultaneously in all three z-, x- and y- axes. Thus, instead of constructing the structure in 2D layers stacked vertically on top of each other, the printer can now utilize more versatile print paths to feasibly create nonplanar structures (and hence the part “Freeform” of FRESH).
Furthermore, since the support material “locks” the bioink in place immediately after extrusion, the printed structure cures in the shape that it was extruded in, and therefore makes the printed structure look nearly identical to the 3D image to the millimeter, and even the micrometer, scale.
How 3D bioprinting works
- First, make a Computer-Aided Design file of the structure you want to print
- Slice the CAD into STL (stereolithography) files
- Run the STL file through software like Cura and slic3r to convert it into a .gcode file, which the printer reads directly from
- Send the .gcode file to the printer
- Let the magic happen ✨
- Slowly heat the print chamber in an incubator overnight and let the support bath wash away
Challenges of Bioprinting
Challenge1 — size of printed structures
Theoretically, the maximum printable size is limited by the printer build volume (the maximum volumetric range in which the nozzle can move) and the volume of bioink available.
Although the former problem is easily fixable by using a larger printer, the latter — finding a way to print with large volumes of bioink — is more complicated. One possible solution is refilling the syringe or replacing it with new, filled ones. This option, however, requires re-aligning the location of the syringe with the x, y, and z axes of the printer for every switching 😕. Another option is to replace the syringe pump with a progressive cavity pump, which draws the print material from a large reservoir.
Regardless of how you choose to store a large volume of bioink, we must also deal with preventing the cells from settling at the bottom within the syringe. Unlike the desirable homogeneous distribution of cells within the bioink, uneven suspension of cells within the ink reservoir would result in cell densities that are concentrated only in certain parts of the printed structure.
Challenge 2 — print time
The balance between print time (= amount of time in which the cells are outside of their optimal environment) and print quality (smoothness and fidelity to the original 3D image) presents a major dilemma that affects all bioprinting situations.
On one hand, decreasing print time by either increasing layer height, decreasing infill density, and increasing movement velocity of the nozzle would lower the print quality and biological property of the printed construct. On the other hand, maximizing print quality (lowering layer height and print velocity to make the structure look smoother) would inevitably lengthen the print time, and increase the risk of cell death during the printing process.
Scientists have tried to keep the cells alive for longer by mixing nutrients into the liquid phase and increasing or decreasing nozzle temperature to keep cells at their optimal temperature or decrease their metabolic activity, respectively. However, further development in both solutions is necessary to establish more reliable ways to maintain high cell viability through longer print durations.
Our future with bioprinting/applications
As the most well-known application, bioprinting has the potential to fabricate functional organs to replace damaged tissues in patients. Currently, available organ donations fulfill only 10% of the global demand, and 17 people die each day waiting for a new organ. Once we accomplish fully functional organ 3D printing, patients could simply have their own organ printed with cells from their own body in a matter of hours!
Printing organs will also expedite our drug screening process by enabling the simulation of the interaction of drug candidates with the human body. This also means we wouldn’t need to sacrifice animals 🐶 to prove the safety of new drugs.
Companies like Organovo, Allevi, and Aspect Biosystems are developing bioinks and bioprinters (including FRESH bioprinters) to take us closer to such a future.
Works Cited:
- Emergence of FRESH 3D printing as a platform for advanced tissue biofabrication (Feinberg et al.) — https://aip.scitation.org/doi/10.1063/5.0032777
- Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels (Feinberg et al.) — https://www.science.org/doi/10.1126/sciadv.1500758
