It was only a matter of time before the iconic scene from 1997’s The Fifth Element became a reality. In the film Milla Jovovich’s character, Leeloo, is entirely rebuilt using her dead hand as a template. The resurrection is performed by a surgical robot that collects slices of heterogeneous tissue, generated from a yellow bio-ink, and places them rapidly in sequence, followed by the addition of softer tissues via long red fibres.
Unfortunately, in the year 2018, this spectacle remains stubbornly science fiction. However, at the same time, almost every human tissue is currently being tested for clinical application, from blood vessels to smooth muscle and neural tissue. Furthermore, a recent product boom in the area of 3D bioprinting is set to accelerate the field of tissue engineering, leading towards the printing of more complex tissues and even full organs.
To give a general introduction to the 3D bioprinting space, this article briefly summarizes noteworthy printing systems, focusing on their distinguishing features. As universities increasingly adopt these systems and find new applications for them, new routes to high-impact research and innovation will unfold in the coming months and years.
A Few Words to Set the Scene
The first thing to note is that 3D bioprinting companies currently share a few intentions. Firstly, all companies claim to closely mimic native tissues. This is the foundation of selling in the bioprinter space: “our system gives the most realistic results”. That means creating tissues that contain multiple cell types and exact replica geometries of tissues found in the body (or in animal bodies). This is, of course, crucially important because these tissues will mostly be used for drug testing or implants. Without going into depth, in summary, using animal models for drug testing is expensive and often unreliable and unethical. As such, many parties, including the U.S. Food and Drug Administration (FDA), see cultured human tissues as a viable alternative to animal testing. To this end, the FDA signed a collaborative agreement with a company, Emulate, in 2017 to harness their Organs-on-a-Chip technology. [Emulate is partnered with Johnson & Johnson and Merck]. Overall, the ability to precisely recreate the in-vivo human experience of a drug is (*overused term alert*) the Holy Grail of tissue culturing as it pertains to drug discovery.
Secondly, all bioprinting companies want to extend the range of materials that are compatible with their systems. To deal with highly-viscous materials that are difficult to push through small channels, heating technology is becoming the norm [insofar as heating lowers viscosity]. Some systems are even including hot plastic-melting tools that work in tandem with cooler bioprinting tools. Thirdly, with huge amounts of innovation in the area of bioprinting, companies are looking to make their systems flexible and future-proof, meaning they prefer a modular design approach wherever possible. Simple things like printing resolution (currently up to 100 microns) will see improvements, but complex changes are also on the horizon, such as new chemical cross-linking techniques, new approaches to scaffold-building and cell-impregnation, and next-generation bio-inks.
With all of that said, let’s take a look at the companies and their latest products:
CELLINK – U.S.A.
The Bio X Bioprinter is another relatively inexpensive option at around CAD$15-20k. The system has three print heads that snap on and off, which can accommodate syringe extruders (with heating/cooling from 4-250 ℃), a UV curing tool or a video camera. This gives a maximum of three materials at a time if only extruders are attached to the print heads. Similar to Allevi, Cellink uses value add-ons to make the system more desirable that simply ‘hacking’ your own 3D bioprinter (see end of article). In the case of Cellink, this means their patented Clean Chamber technology, which uses HEPA filters, UV-C germicidal control (for sterilization cycles) and positive air pressure inside chamber to maintain a pristine work space. Their BIO X6 bioprinter is available to let users print with 6 printheads at a time.
Advanced Solutions – USA
The BioAssemblyBot® is the first of its kind – a 3D bioprinter mixed with a robotic arm. This six-axis robot is highly-manoeuvrable and can hold a variety of tools, including syringe extruders and a video camera for monitoring the process [extruders can be heated/cooled in the range 5-110 ℃]. These tools are interchangeable, making the system partly modular. It also comes with a significant price tag of around CAD$200-220k. Advanced Solutions also offers a more standard printer, the BioBot, which has a “turret” dispensing head that holds up to five syringe extruders at a time, allowing for print jobs that require up to five different materials.
Allevi – USA
Allevi specializes in compact systems that fit in any fume hood. The latest Allevi 6 has six temperature-controlled syringe extruders (4-200 ℃) supported by light sources (UV and Visible) for curing/cross-linking printed material. [The high temperature range allows printing of thermoplastics]. The system is small and jazzy – it isn’t the most sophisticated printer, but Allevi tries to give customers a big “bang for their buck” with added value items such as wireless control, extruder auto-calibration (standard in more expensive models) and extrusion pressure up to 120 PSI (allowing a wide range of viscosities). Allevi 6 is a relatively inexpensive option at around CAD$15-20k.
Aspect Biosystems – Canada
Many bioprinters use syringe-based extrusion. Not the the RX1™ Bioprinter! This system uses microfluidic chips instead of syringes. The microfluidic channels contain pneumatic valves that allow you to change and mix materials on-the-fly during printing. This capability streamlines the printing process by removing time-consuming steps (i.e. pre-mixing bio-inks; swapping syringes) so print time is only dependent on print volume. On-the-fly mixing paves the way for the RX1’s chemical cross-linking and the formation of cell-laden microfibres using coaxial flow focusing. This coaxial technique is also useful for generating hollow fibres, tissue barrier models, vasculature and tissue co-culture. Overall, it’s an innovative solution to the problem of creating 3D heterogeneous tissues and is part of Aspect’s portfolio of patented Lab-on-a-Printer technology.
Cyfuse Biomedical – Japan
The Regenova does not use scaffolds, just cells! Instead of printing materials, the system arranges cells using micro needle arrays. Cell aggregates (a.k.a. spheroids) are selected, picked up and skewered onto long, 170 micrometre-wide needles, like a shish kebab. Over the course of a few days, the spheroids naturally fuse together to form a continuous tissue. The process of de-cannulation involves removing the early tissue from the needle array, which is then matured to form the final product. The system can be automated to select a wide variety of cell types and plant them at specific locations in the array, giving rise to 3D heterogeneous tissues. However, with such sophistication comes high cost, and the Regenova is estimated at over CAD$320k. In addition, some questions remains as to the speed and risk of cell death compared other techniques.
GeSim – Germany
The BioScaffolder combines three capabilities: 3D printing, electrospinning and pipetting. This allows the system to print or electrospin micro-scale fibres, which make up a scaffold, and then pipette small quantities (down to nanolitres) of low-viscosity material onto the scaffold. The pipetted material can be solutions of cells, proteins or drugs. The system has three extruders for sequential printing of different materials and also includes the latest innovations, namely heating/cooling (0-250 ℃), an FDM (fused deposition modeling) extruder to print commercial filaments, and coaxial extrusion to form hollow fibres, etc. The ability to co-print multiple materials at the same time as high-temperature thermoplastics (using the FDM extruder) is a recent addition to the bioprinting space. For this multi-function printer, customers will be paying around CAD$200-220k.
Organovo – USA
Organovo was the first bioprinting company, launching in 2007. They are currently focused on selling tissues and not bioprinters, but since they advertise their systems we can only assume that might change in the future. Their latest non-product, the NovoGen Bioprinter® Platform, is a temperature-controlled, syringe-based extruder that uses UV curing. This may sound basic (and it is) but Organovo has patents on innovative coaxial nozzles that extrude multiple layers simultaneously. If it’s two layers (core and mantle), it’s possible to achieve previously noted structures, like hollow fibres and vasculature. But Organovo takes this further, with the introduction of one or multiple intermediate layers between the core and mantle, with the extrusion of each layer being controlled independently. This could give rise to interesting structures, for example using layers of sacrificial material (removed after printing).
RegenHU – Switzerland
Similar to GeSim’s BioScaffolder, the 3DDiscovery™ Evolution combines 3D printing and electrospinning. However, instead of adding pipetting to the mix, it adds a completely different dimension: biostimulation. With the 3DDiscovery™ Evolution, you can print micro-scale fibres, electrospin nano-scale fibres and then stimulate the created tissues with mechanical, electrical, hydrodynamic and optical tools. The system uses syringe extrusion and has a total of 11 possible tools, including coaxial extruders (for printing hollow fibres, etc.), heated/cooled extruders (0-200 ℃), and light sources (UV, visible and laser) for curing. RegenHu’s motivation is to mimic nature by allowing users to create nanostructures within microstructures, bring them to life and then stimulate and test them. It’s a far cry from the most basic extrusion printers, costing upwards of CAD$260k and measuring about the size of a small fumehood.
Finally, we have some honourable mentions. These systems are valiant additions to the bioprinting space, but don’t advertise many distinguishing features.
Rokit’s Invivo (South Korea) is a less expensive system (around CAD$50k) using syringe extrusion. It’s temperature control is surprising (-10 to 80 ℃ standard, optional tool goes up to 350 ℃) and it also includes wireless control and the possibility for both UV and chemical cross-linking. Two more systems in the middle price range (CAD$25-50k) are 3Dynamic Systems’ Omega (United Kingdom) and Bio3D’s SYN^ (Singapore), based on syringe extrusion. Lastly, EnvisionTEC’s 3D Bioplotter (Germany) is a syringe-based system with temperature control (-10 to 250 ℃), UV curing and a sterile biosafety working space that meets requirements for clinical trials. The system is one of the more tried-and-tested on the market and goes for over CAD$250k.
Hack Your Own Bioprinter
If you want to create scaffolds and haven’t secured funding for one (or more) of the printing systems described above, you may want to consider “hacking” an ordinary 3D printer. The basic steps are outlined here (performed by a group from Carnegie Mellon University), which boil down to rigging the printer with syringe extrusion capability.
However, there are many downsides to performing expensive research in this way. First of all, you’ll have to factor in the cost of the 3D printer and the time spent adapting it. Secondly, the system won’t have any bio-related functions, such as temperature control, light/chemical curing or multiple extruders. Thirdly, the system won’t be optimized to reduce shear forces that may lead to cell death, potentially costing money down the line. And, finally, you won’t have any support, which is a primary function of the big companies currently operating in the bioprinting space. Ultimately, if you’re going to be using the system for high-impact research, probably better to go for the real deal.