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A Guide to Bioprinting: Understanding a Booming Industry

The success of bioprinting could become the key enabler that personalized medicine, tissue engineering, and regenerative medicine need to become a part of medical arsenals. Breakthroughs in bioprinting will enable faster and more efficient patient care and recovery. Biofabrication could be used to reshape the foundations of drug development, medicine, cosmetics, organ transplantation, and many other fields. It will transform the way doctors repair damaged ligaments, recreate tissues, and even reproduce the layers of the skin.

We are entering an era of bioprinting revolution. But to understand the role that bioprinting will play in the future, it is important to look back at how early discoveries in the field provided a strong basis to push its capability forward:

Back in the ’90s, Anthony Atala, pediatric surgeon, urologist, and director of the Wake Forest Institute for Regenerative Medicine (WFIRM) in North Carolina, created by hand bladders, skin, cartilage, urethra, muscle, and vaginal organs. By the end of that decade, the Institute used a 3D printer to build a synthetic scaffold of a human bladder, which they then coated with cells taken from their patients and implanted it, preparing the stage for bioprinting.
Then in 2002, scientists from Harvard University printed two-inch-long mini-kidneys capable of filtering blood that was then transplanted back into genetically identical cows, where they started making urine. The novel research raised the prospect of using stem cells taken from human patients with kidney failure to create new organs for transplant.
However, it wasn’t until 2003, when bioengineering professor Thomas Boland adapted a Hewlett-Packard inkjet printer in his lab at Clemson University to begin printing a bioink made of living bovine cells suspended in the cell-culture medium, that bioprinting began to materialize. This led to the creation of the world’s first 3D bioprinter, capable of creating living tissue from a solution of cells, nutrients, and other bio-compatible substances.

picture os a man using the bioprinter on a limb

A member of WFIRM team operates with the bioprinter (Image: WFIRM)

Twenty years later, researchers still face challenges as they continue working with bioprinters and bioinks. Even though there has been an increasing adoption of the technology, the extent of its potential has not been fully exploited. From choosing the bioinks to actually bioprinting human tissues and organs, this new field is quickly becoming the go-to technology that bioengineers, researchers, and hospitals need to evolve from lengthy and cumbersome manual work to scalable and replicable results.

How Well Do you Know Bioprinters? (or How Bioprinters Work and Who Makes Them)

Bioprinters work by extruding cells and other biomaterials contained in bioinks, from syringes that deposit the material layer by layer to create different types of tissues or organ-like constructs. The technology behind the bioprinters vary. Nonetheless, to date, the three main and most popular bioprinting technologies are extrusion, inkjet, and laser-based bioprinting. Some mainstream examples are:

Some manufacturers, like Cellink or Allevi, use pneumatic-driven extrusion systems that pump high-pressure air in a cartridge to force bioinks to flow through a nozzle.
Other fabrication systems, such as the one designed by Poietis has laser-assisted bioprinting that allows cells to be positioned in three dimensions with micrometric resolution and precision to design living tissue.
Another type of bioprinting technology uses a stereolithography-based bioprinting platform. Vendors using this process include Volumetric and Cellink’s jointly produced Lumen X projection stereolithography based bioprinter.
Another project that could revolutionize the way surgical procedures are performed is handheld bioprinters; these systems enable surgeons to deploy cells — or material to aid in cellular growth — directly into a defect site in the body, such as severely burnt skin, corneal ulcerations or bone. One of the most talked-about handheld bioprinters has been Australia’s University of Wollongong BioPen, allow surgeons to repair damaged bone and cartilage by “drawing” new cells directly onto bone in the middle of a surgical procedure. Although still in pre-clinical trials, these devices have attracted the attention of healthcare practitioners due to its versatility.

A few of the main manufacturers supplying the market include 3D Bioprinting Solutions, Allevi, Aspect Biosystems, Cellink, nScrypt, regenHu, Inventia, Regemat3D, Poietis, and more. Last year, 3DPrint.com counted 111 established bioprinting firms around the world. Mapping the companies that make up this industry is a good starting point to understand the bioprinting ecosystem, determine where most companies have established their headquarters and learn more about potential hubs, like the one in San Francisco.

Types of Bioinks

3D bioprinters use bioinks. Bioinks are substances made of living cells that can be used for 3D printing complex tissue models — they mimic an extracellular matrix environment to support the adhesion, proliferation, and differentiation of living cells. Choosing which bioink to use can be challenging. To date, we have witnessed researchers using bioinks based on several biomaterials, such as alginate, gelatin, collagen, silk, hyaluronic acid, even some synthetic-biomaterials-based-bioinks.

The promise of hydrogels. A macromolecular polymer gel constructed of a network of cross-linked polymer chains, hydrogels are able to meet the stringent requirements of cells and are the basis of almost all bioink formulations. As stated in “Engineering Hydrogels for Biofabrication”, published in Advanced Materials, “hydrogels are particularly attractive for biofabrication as they recapitulate several features of the natural extracellular matrix and allow cell encapsulation in a highly hydrated mechanically supportive three‐dimensional environment.” This makes hydrogel-based bioinks a very promising choice for many researchers and bioengineers.

Bioinks from patients’ cells. The biomaterials can also use a patient’s own cells, adult stem cells, manipulating them to recreate the required tissue. The source of the cells varies depending on what researchers are bioprinting. For example, in the case of Alzheimer’s disease, experts at the University of Victoria in Canada, have bioprinted neural tissues using stem cells as a tool for screening drug targets for the disease. The ability to program patient-specific cells is the beginning of customized bioprinting since the unlimited potential of these cells can be used to regenerate or repair damaged tissue.

Polbionica’s bioinks (Image: Polbionica)

What is Bioprinting Good For?

What is most exciting about bioprinting, are the many ways that doctors and researchers are using currently available devices in the market or are creating their own systems to facilitate new processes and applications. The orchestrated interaction between machine and user has led to innovation that could reinvent the world of tissue engineering.

Moving oncology forward: Bioprinting is being employed in the battle against cancer, whereby scientists create tumor models for research. Modeling cancer using 2D cell cultures fails to accurately replicate the microenvironment of tumors. This is why scientists have turned to biofabrication tools to make three-dimensional models that mimic the intricate in vivo tumors. These models help test anticancer drugs; aid scientists in understanding the underlying causes of metastasis, and can even personalize treatment for individual cancer patients. There have been plenty of initiatives that apply bioprinting to oncology. These range from immersion bioprinting of human organoids to printing cancer tissues in 3D.

Microtumors (Image: CTI Biotech)

The market for oncology-oriented bioprinting seems sure to grow. The number of patients suffering from the disease continues to go up. In 2018 alone there were 17 million new cases of cancer worldwide, and projections suggest that there will be 27.5 million new cases of cancer each year by 2040. What that effectively means is that we are witnessing an increase in oncology bioengineering research and whether it is for glioblastoma, bone cancer tumors, or lung models with tumors, the implications can be profound since the ability to use bioinks and bioprinters to create tumors frees researchers of the many ethical concerns associated with testing as well as reduces the costs associated with such research activities.

Building scaffolds: Probably the most important practical use for bioprinting at the present time is in regenerative medicine. For instance, in 2019, researchers from North Carolina State University (NCSU) and the University of North Carolina at Chapel Hill created a 3D biomedical fiber printer used to create biocompatible scaffolds. Also, Harvard researchers working in Jennifer Lewis’ Lab at Harvard´s Wyss Institute for Biologically Inspired Engineering and John A. Paulson School of Engineering and Applied Sciences (SEAS), came up with a much talked about breakthrough new technique, the SWIFT method, that allows 3D printing to focus on creating the vessels necessary to support a living tissue construct. A team of researchers at Texas A&M University have even developed a 3D printable hydrogel bioink containing mineral nanoparticles that can deliver protein therapeutics to control cell behavior.

Limitations of Bioprinting

Though we heard it many times before, knowing that someday 3D printed artificial organs could eliminate the need for an organ donor waiting list is comforting. Creating personalized replacement organs sounds like the solve-all solution to the organ shortage crisis, yet, a functional organ compatible for human implantation may be decades away. Today, 3D printed organs are still raw to be used for transplantation and lack the vasculature required to function within the human body.

Creation. Last year, mainstream news outlets headlined a story about researchers who had 3D printed a heart. However, the published scientific paper behind that story described how a group of scientists from Tel Aviv, Israel, created bioink out of heart cells and other materials from a patient, and were able to develop cardiac patches and ultimately, 3D print comprehensive tissue structures that include whole hearts. The tissue was shaped in the form of a tiny heart that was kept alive in a nutrient solution. The paper expresses how this development could not function like a real heart since the cells in the construct can contract, but don’t yet have the ability to pump.

This was certainly not the first heart to be 3D printed, yet Tal Dvir, who led the project at Tel Aviv University’s School of Molecular Cell Biology and Biotechnology, indicated that never before had it resulted in an organ “with cells or with blood vessels.” It was an amazing breakthrough for the field, and it proves that biotechnology has made significant advances, but it is still a long way from creating organs that can be transplanted to people, considering that the vasculature — the network of blood vessels that feeds the organ — remains a challenge, but scientists are determined to troubleshoot these issues.

So, no matter how enticing the idea of successfully bioprinted organs sound, stories like this remind us to keep the hype in check, making the work of news outlets fundamental for reporting advancements in research and medical breakthroughs (which usually take much more time).

Where to Next?

Organ bioprinting. The application of 3D bioprinting will be a game-changer in medicine, as the machines successfully replicate tissues and organs, build muscles and cartilage, and enable the adoption of customized medicine. The long-term dream for bioprinting has always been the routine printing of body organs. Current ongoing projects include Michal Wszola’s 3D bioprinted bionic pancreas or Organovo’s 3D bioprinted liver.

The space frontier. Bioprinting in space could hold the key to developing fully functional organs. This is because bioprinting without gravity allows organs to grow without the need for scaffolds. The National Aeronautics and Space Administration (NASA) considers that terrestrial gravitation represents a significant limitation, while a gravity-free environment, magnetic and diamagnetic levitation will allow for biofabrication of 3D tissue constructs with a scaffold-free and even nozzle-free approach. Some bioprinters have already been launched and used in space. This includes nScrypt and Techshot’s BioFabrication Facility (BFF), or the Organaut 3D bioprinter created by Russian biotech firm 3D Bioprinting Solutions and Roscosmos, the Russian state corporation responsible for space flights.

Healthcare at its best. More prosaically, biomaterials specialist and a professor of biofabrication at Queensland University of Technology, Australia, Mia Woodruff, has been advocating the hospitals of the future for years. She has an exciting vision of a future where the fabrication of patient-specific replacement tissue and organs is safe, cost-effective, and routine. Though perhaps years from happening, her vision is in tune with what many think bioprinting could become, that is, with enough researchers, companies, and funding.

An astronaut aboard the ISS using Techshot’s BioFabrication Facility or BFF (Image: Techshot/NASA)

Coming regulation. Back here on Earth, there will be a growing need for common guidelines for bioprinting to make the process more standardized. In the EU, for example, there currently no particular regulatory regime governing the whole bioprinting process, but piecemeal legislation is relevant in relation to tissue engineering and regenerative medicine. While the Food and Drug Administration (FDA) of the United States plans to review the regulatory issues related to the bioprinting of biological, cellular and tissue-based products in order to determine whether additional guidance is needed beyond the recently released regulatory framework on regenerative medicine medical products.

As the development of the technology strongly advances and proves successful for researchers, we will surely continue to observe brilliant minds perfecting devices and biomaterials, envisioning new systems for future needs, especially as startups emerge out of universities and research institutes, and established companies upgrade their machines to face the limitations we previously addressed in this article.

Still, there is a long way to go, what was largely built so far is a very promising technology. For instance, fully functional organ fabrication for transplantation might take decades. Nonetheless, the unquestionable contribution of bioprinting to so many fields remains an incentive to invest in this area to overcome medical challenges and to move the healthcare industry in a different path, where technology will not only aid in curing diseases but also guiding people by helping them stay healthy, recognizing symptoms early and personalizing solutions in real-time.

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Indianapolis VA Medical Center and NASA To Explore 3D Bioprinting for Healthcare

Exposure to space radiation, gravity fields, contaminated atmospheres, are all part of the hostile environment astronauts encounter beyond Earth’s orbit. As they transition from one gravity field to another, the fluids in their body can shift upwards, which could lead to pressure on the eyes causing vision problems, among other pretty scary diseases that the National Aeronautics and Space Administration (NASA) has been working to solve for decades as they prepare to send humans on three-year missions to Mars.

NASA wants to make sure that tissue replacement beyond the repairing ability of the organism can be done in space. However, such a scheme would also mean that space agencies will have to plan way ahead of departure on how to leverage innovative technologies, like 3D printing, to aid astronauts on their journey, while directly contributing to their respective development efforts. That is what several United States government agencies are doing, including the Veterans Administration and NASA. More particularly, scientists at the Indianapolis Richard L. Roudebush Veterans Affairs (VA) Medical Center and NASA investigators are now jointly exploring new approaches to 3D bioprinting. The two teams are collaborating in the conversion of human and animal eye fundus images into virtual renderings of the retinal vascular networks for bioprinting. Exploring this field, will not only serve NASA’s interest in space, but also the VA’s concerns here on Earth since tissue replacement is often needed to treat combat, accident or disease-induced damage to veterans.

One of the most serious limitations for the success of tissue engineering, and in particular of the bioprinting approaches to generate artificial tissues, is the difficulty to perfuse the constructs with oxygenated fluids and nutrients. To tackle this issue, Patricia Parsons-Wingerter, senior scientist at NASA’s Space Biosciences Research Branch, and her NASA-wide team joined forces with Nicanor I. Moldovan, founding director of the 3D Bioprinting Core (3DBPC) laboratory at the Richard L. Roudebush VA Medical Center.

Patricia Parsons-Wingerter (Credit: NASA)

Such combined efforts could bring unprecedented success for the bioprinting of vascular networks. In this instance, the NASA team will use their innovative Vessel Generation Analysis (VESGEN) 2D software application for the design of anatomically realistic vascular patterns for bioprinting, while the Indianapolis-based team will provide their know-how on bioprinting vascular models.

In fact, Parsons-Wingerter and her colleagues developed VESGEN 2D, an image analysis program that performs branching quantification of vascular networks, in order to assist in the early detection of changes in vascular patterns of the eye, which may indicate microgravity-induced retinopathy, also the major blinding disease of working-aged adults. However, vascular-dependent diseases include cancer, diabetes, coronary vessel disease, and major astronaut health challenges in the space microgravity and radiation environments, especially for long-duration missions. In this sense, VESGEN 2D maps and quantifies vascular remodeling for a wide variety of quasi-2D vascularized biomedical tissue applications

Parsons-Wingerter’s concern with eyesight problems related to space travel has been historically well-founded. According to one study sponsored by NASA several years ago, space flights that last six months or longer can cause changes in astronauts’ eyes and vision. At the time, this discovery had a major impact on plans for a manned flight to Mars, making this a top priority for NASA’s Space Medicine research team. During the study, seven astronauts that were examined had eye structure and vision abnormalities, most commonly the flattening of the back of the eyeball, changes in the retina (the light-sensitive area at the back of the eye) as well as the optic nerve. Knowing now that vision can be severely compromised in space now demands a lot of attention from researchers, especially, since astronauts, like airline pilots, must have 20/20 vision to carry out most of their work in orbit.

According to Parsons-Wingerter, VESGEN is in fact, currently being used to help understand and ameliorate vision impairments in astronauts and terrestrial adults diagnosed with diabetic retinopathy. And it’s no surprise that VESGEN 2D will be utilized to aid in the design of anatomically realistic vascular patterns for bioprinting since this image analysis program offers insightful read-outs (or biomarkers) of dominant molecular signaling targeted by drug and therapeutic development.

Composite images and journal cover illustrations of vascular patterning collaborations with other scientists for VESGEN research (Credit: NASA)

Meanwhile, Moldovan’s 3DBPC laboratory (also known to many as the “Core”) primarily serves the VA investigators’ needs, helping with workflow design and implementation from project conception through funding and execution. More specifically, the Core provides assistance with hydrogels preparation, bioinks characterization, cell cultivation, samples mixing, 3D model printing, cell-containing constructs bioprinting, incubation, perfusion, and characterization (by fluorescence microscopy, micro, and macro photography), up to data analysis and preparation for publication. Current activities in the Core include the generation of bone, cartilage, retinal, neural, and other 3D models.

Nicanor Moldovan (Credit: 3D Tissue Bioprinting Core at VAMC)

Funded and administered by the Indiana Institute of Medical Research (IIMR) and supporting the overall research mission of the Veterans Affairs Medical Center in Indianapolis, the Core features the bioprinter 3DDiscovery , which was purchased from Swiss company regenHU through a VA Shared Equipment Evaluation Program (ShEEP) grant.

Another example of the commitment of government agencies to work together to develop healthcare solutions for the future, the Richard L. Roudebush VA Medical Center has also recently joined NASA’s centennial Vascular Tissue Challenge, a project that rallies scientists worldwide to produce viable thick-tissue assays that can be used to advance research on human physiology. NASA began the Vascular Tissue Challenge in May 2019, in collaboration with the Methuselah Foundation and thanks to the support from the New Organ Alliance (NOA), a non-profit organization aiming to catalyze the tissue engineering field. Roudebush VA Medical Center’s Moldovan is also participating in this venture as chair of the NOA’s In Vitro Tissue Models Sub-Committee.

Considering that one of the main projects in the Core is the bioprinting of vascular models, this new joint effort with Parsons-Wingerter is considered by them as a natural step to move forward the research for both teams. In fact, the same approach with VESGEN 2D can be used for the realistic representation, and the adaptation to the printable format of vascular networks of other organs, such as rat mesentery or mouse colon, to be incorporated as region-specific cellular compositions in actual bioprinted tissue constructs. The teams claim that these constructs will be useful both as in vitro models for mechanistic studies and drug discovery and for the eventual replacement of damaged tissues or organs. After all, that is exactly what NASA and the VA are aiming for, both here and in space.

Considering that the US space agency has recently asserted its goal of sending astronauts to Mars once again, researchers, like Parsons-Wingerter and Moldovan, are crucial to the future mission’s, success. This is especially relevant since such Mars missions would last from seven months to two or more years. As they continue to solve many of the health-related issues attached to long-duration space expeditions, we will surely find out more about their work as they combine creative talents to develop more bioprinting innovation.

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Techshot’s Bioprinter Successfully Fabricates Human Menisci in Space

Current Bioprinting in space could become a pathway that guides future decisions for biofabrication on Earth as well as in orbit. Astronauts have already used two bioprinters on the International Space Station (ISS), experimenting with human bone tissue and even heart tissue. Interest in creating these machines arose as Earth’s gravity was making printing functional organ-like structures quite challenging, making the space environment a feasible alternative. Techshot, a commercial space company, chose to develop a BioFabrication Facility (BFF) that has been mounted inside the ISS U.S. National Laboratory and is being used by astronauts on board since last summer. This week the company announced that the space-based 3D bioprinter was used to successfully manufacture human knee cartilage test prints in space.

Techshot’s BFF, which aims to print organ-like tissues that could one day lead to 3D printing human organs in space for transplants, was used to successfully manufacture test prints of a partial human meniscus aboard the ISS last month. The meniscus pattern was manufactured for the company’s customer: the 4D Bioprinting, Biofabrication, and Biomanufacturing (4D Bio³) program, which is based at the Uniformed Services University of the Health Sciences (USU). The program is a collaboration between the university and The Geneva Foundation, a non-profit organization that advances military medical research.

BioFabrication Facility Patch (Image: Techshot)

Manufacturing human tissue in the microgravity conditions of space could ultimately aid in the race to manufacture hearts and other organs using a 3D bioprinter. Although the actual fabrication of functional organs that could finally replace the shortage of donor organs to help patients in need of a transplant could be a decade away – if not more – the team at Techshot was optimistic around this project since research in space might illuminate a lot of the work done on Earth.

In the last six months, astronauts, like NASA’s flight engineer Christina Koch, have tested the ability of the BFF to print cells. Using adult human cells (such as stem or pluripotent cells) and adult tissue-derived proteins as its bioink, the BFF is able to create viable tissue.

Astronaut Jessica Meir is using the BFF at the International Space Station (Image: Techshot)

According to the ISS U.S. National Lab, although researchers have had some success with 3D printing of bones and cartilage on Earth, the manufacturing of soft human tissue (such as blood vessels and muscle) has been difficult. What they claim occurs is that, on Earth, when attempting to print with soft, easily flowing biomaterials, tissues collapse under their own weight, resulting in little more than a puddle; but if these same materials are produced in the microgravity environment of space, the 3D printed structures will keep their shapes.

A meniscus, which is a crescent-shaped disc of soft cartilage that sits between the femur and the tibia, acts as a significant cushion or shock absorber, yet when the meniscus tears, the cushioning effect functions poorly, leading to arthritis and knee pain. Meniscal injuries are one of the most commonly treated orthopedic injuries and have a much higher incidence in military service members and sports players.

Early in March, Techshot sent equipment and samples supporting plant, heart and cartilage research for three of its customers to the ISS on SpaceX mission CRS-20. Astronauts on-board the station used the BFF to manufacture human knee menisci as a test of the materials and the processes required to print a meniscus in space. According to Techshot, the first experiment for 4D Bio³ aboard the ISS U.S. National Laboratory served as a test of the materials and the processes required to print a meniscus in space. Astronaut Andrew Morgan, a medical doctor and graduate of USU loaded biomaterials into BFF, while Techshot engineers uploaded a customer-provided design file to the printer from the company’s Payload Operations Control Center (POCC) located in Greenville, Indiana, from which the devices in space are controlled. The success of the print was evaluated via real-time video from inside the unit.

“Some of our criteria for mission success, such as the ability to work with customer-specified print materials and customer-supplied design files, were met before we even launched back on March 6,” said Techshot Senior Scientist Carlos Chang. “But commanding BFF to print from here at Techshot, and watching it all literally come together in real-time, provided the confirmation we needed that we’re on the right track.”

Founded more than 30 years ago, Techshot operates its own commercial research equipment in space and serves as the manager of NASA-owned ISS payloads – such as the Advanced Plant Habitat and two materials-science research furnaces. The company provides its catalog of equipment and services for a fee to those with their own independent research programs – serving as a one-stop resource for organizations seeking access to space. And launched to the station in July 2019 aboard SpaceX CRS-18, the BFF has been tested since. Techshot has even suggested that biomaterials for a second meniscus print, which will be returned to Earth for more extensive testing, will launch on a later SpaceX mission.

As astronauts stationed at the ISS U.S. National Lab continue to advance work with Techshot’s 3D bioprinter and microgravity research, we can expect to hear more about the cutting edge science that is being done that aims to improve patient care. The technology offers a unique opportunity to support bioprinting structures and construct tissues, providing an ideal scenario that will enable remarkable changes to move forth the medicine of the future.

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Improvements to the BioFabrication Facility on the ISS Thanks to Lithoz

Scientific discoveries and research missions beyond Earth’s surface are quickly moving forward. Advancements in the fields of research, space medicine, life, and physical sciences, are taking advantage of the effects of microgravity to find solutions to some big problems here on Earth. Researchers in 3D printing and bioprinting have taken advantage of space facilities that are dedicated to conducting multiple experiments in orbit, such as investigating microgravity’s effects on the growth of three-dimensional, human-like tissues, creating high-quality protein crystals that will help scientists develop more effective drugs, and even growing meat with 3D printing technology.

The BioFabrication Facility (BFF) by Techshot and nScrypt (Credit: Techshot)

On November 2, 2019, a Northrop Grumman Antares rocket successfully launched a Cygnus cargo spacecraft on a mission to the International Space Station (ISS). The payload aboard the Cygnus included supplies for the 3D BioFabrication Facility (BFF), like human cells, bioinks, as well as new 3D printed ceramic fluid manifolds that replaced the previously used that were printed out of polymers. According to Lithoz – the company behind the 3D printed ceramic fluid manifolds – they are enabling advancements in bioprinting at the ISS.

The additive manufactured ceramics have been in service since November 2019 and Lithoz claims they have proven to provide better biocompatibility than printed polymers, resulting in larger viable structures.

Lithoz, a company specializing in the development and production of materials and AM systems for 3D printing of bone replacements and high-performance ceramics, printed the ceramic manifolds using lithography-based ceramic manufacturing (LCM) on a high-resolution CeraFab printer in collaboration with Techshot, one of the companies behind the development of the BFF. Moreover, the ceramic fluid manifolds are used inside bioreactors to provide nutrients to living materials in space by the BFF.

Testing of the ceramic 3D printed manifolds is focusing on biocompatibility, precision, durability, and overall fluid flow properties; and the latest round of microgravity bioprinting in December yielded larger biological constructs than the first BFF attempts in July.

NASA engineer Christina Koch works with the BioFabrication Facility in orbit (Credit: NASA)

Techshot and Lithoz engineers and scientists worked together to optimize the design and the manufacturing processes required to make it. Techshot Senior Scientist Carlos Chang reported that “it’s been an absolute pleasure working with Lithoz.”

While Lithoz Vice President Shawn Allan suggested that “their expertise in ceramic processing really made these parts happen. The success of ceramic additive manufacturing depends on working together with design, materials, and printing. Design for ceramic additive manufacturing principles was used along with print parameter control to achieve Techshot’s complex fluid-handling design with the confidence needed to use the components on ISS.”

Headquartered in Vienna, Austria, and founded in 2011, Lithoz offers applications and material development to its customers in cooperation with renowned research institutes all over the world, benefiting from a variety of materials ranging from alumina, zirconia, silicon nitride, silica-based for casting-core applications through medical-grade bioceramics.

This work, in particular, highlighted an ideal use case for ceramic additive manufacturing to enable the production of a special compact device that could not be produced without additive manufacturing while enabling a level of bio-compatibility and strength not achievable with printable polymers. Lithoz reported that Techshot engineers were able to interface the larger bio-structures with the Lithoz-printed ceramic manifolds and that the next steps will focus on optimized integration of these components and longer culturing of the printed biological materials. While conditioned human tissues from this mission are expected to return to Earth in early 2020 for evaluation.

Back in July 2019, Gene Boland, chief scientist at Techshot, and Ken Church, chief executive officer at nScrypt, discussed the BFF at NASA’s Kennedy Space Center in Port Canaveral, Florida, how they planned to use the BFF in orbit to print cells (extracellular matrices), grow them and have them mature enough so that when they return to Earth researchers can encounter a close to full cardiac strength. Church described how a tissue of this size has never been grown here on Earth, let alone in microgravity. The 3D BFF is the first-ever 3D printer capable of manufacturing human tissue in the microgravity condition of space. Utilizing adult human cells (such as pluripotent or stem cells), the BFF can create viable tissue in space through a technology that enables it to precisely place and build ultra-fine layers of bioink – layers that may be several times smaller than the width of a human hair – involving the smallest print nozzles in existence.

Flight engineer Andrew Morgan works with the BioFabrication Facility (Credit: NASA)

Experts suggest that bioprinting without gravity eliminates the risk of collapse, enabling organs to grow without the need for scaffolds, offering a great alternative to some of the biggest medical challenges, like supplying bioprinted organs, providing a solution to the shortage of organs.

With NASA becoming more committed to stimulating the economy in low-Earth orbit (LEO), as well as opening up the ISS research lab to scientific investigations and experiments, we can expect to learn more about some of the most interesting discoveries that could take place 220 miles above Earth. There are already quite a few bioprinting experiments taking place on the ISS, including Allevi and Made In Space’s existing Additive Manufacturing Facility on the ISS, the ZeroG bio-extruder which allow scientists on the Allevi platform to simultaneously run experiments both on the ground and in space to observe biological differences that occur with and without gravity, and CELLINK‘s collaboration with Made In Space to identify 3D bioprinting development opportunities for the ISS as well as for future off-world platforms. All of these approaches are expected to have an impact on the future of medicine on Earth.

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The Future of Bioprinting Research Has a New Road Map

Improving efficiency, optimizing technology, increasing awareness, even reducing costs and time, these are all traits that result from strategic road maps, and in the case of bioprinting, where the outcomes affect tissue engineering, bespoke outcomes for patients, regenerative therapy, and much more, having a blueprint for the entire industry seems like a bright idea. Especially when this blueprint highlights some of the challenges on the way to achieving meaningful and innovative scientific development.

Published in IOP Science’s Biofabrication Journal, “The Bioprinting Roadmap” features advances in selected applications of bioprinting and highlights the status of current developments and challenges, as well as envisioned advances in science and technology. The roadmap brings together researchers, specialists, and physicians from a myriad of universities, institutions, and hospitals around the world, combining their knowledge to focus on different aspects of bioprinting technology. These include: Jürgen Groll, professor of functional materials in medicine and dentistry at the University of Würzburg in Germany; Binil Starly, professor of mechanical engineering at North Carolina State University; Andrew Daly, an orthopedic surgeon at Emory University Hospital; Jason Burdick, a bioengineer from the University of Pennsylvania; Gregor Skeldon, a life science medical writer at Maverex, in the UK; Wenmiao Shu, professor of biomedical engineering at the University of Strathclyde in Glasgow; Dong-woo Cho, mechanical engineer at Pohang University of Science and Technology; Vladimir A. Mironov, chief scientific officer of 3D Bioprinting Solutions, and many more.

The roadmap focuses on a broad spectrum of topics, in a detailed and readable fashion that showcases broad knowledge of the field by its authors, as well as a great deal of research that went into the making of the guide. The paper is categorized into the following sections:

Charting the progress from cell expansion to 3D cell printing
Examining the developments and challenges in the bioinks used for bioprinting
Looking into bioprinting of stem cells
Presenting a strategy for bioprinting of tissue vascular systems and tissue assembly:
Examining the potential for using 3D printed biohybrid tissues as in vitro biological models for studying disease
Analyzing how 3D bioprinting can be used for the development of organs-on-a-chip
Biomanufacturing of multi-cellular engineered living systems
Exploring how researchers are pushing boundaries with bioprinting in space
Investigating the developments of bioprinting technologies

The introduction of the research work was in charge of Wei Sun, chair professor of the College of Engineering from Drexel University, in Philadelphia, and Tsinghua University in Beijing, China, who said to IOP Publishing that “there are a number of challenges to overcome, including the need for a new generation of novel bioinks with multi-functional properties to better transport, protect and grow cells during and after printing; better printing processes and printers to deliver cells with high survivability and high precision; efficient and effective crosslinking techniques and crosslinkers to maintain the structure integrity and stability after printing; integration with microfluidic devices to provide a long term and a simulated physiological environment to culture printed models.”

Wei Sun (Credit: Drexel University)

“Due to the rapid advancements in bioprinting techniques and their wide-ranging applications, the direction in which the field should advance is still evolving,” went on Sun. “The roadmap aims to address this unmet need by providing a comprehensive summary and recommendations, useful to experienced researchers and newcomers to the field alike.”

The research sheds light on the main roadblocks to overcome in the future. The specialists consider that the next technologies will use “multiple modalities” in one single platform and “novel processes, such as cell aggregate bioprinting techniques” to create scalable, structurally‐stable, and perfusable tissue constructs. But in the meantime the challenges that remain are many. In his introduction, Sun talks about the need for a new generation of novel bioninks with multifunctional properties to better transport, protect, and grow cells during and after printing; better printing processes and printers to deliver cells with high survivability and high precision; efficient and effective crosslinking techniques and crosslinkers to maintain bioink structural integrity and stability after printing, and integration with microfluidic devices to provide a long-term and a simulated physiological environment in which to culture printed models.

Other researchers highlighted the importance of further improving bioreactor-based cell-expansion systems to lower barriers to the adoption of bioprinting in regenerative medicine and tissue engineering product markets. While Skeldon and Shu suggest that stem cell bioprinting can be realized if scientists find a way to reduce the shear stress of bioprinting stem cells.

Another obstruction is the high production costs and the difficulty of having large-scale production of organoids. Jinah Jang and Dong-Woo Cho from the Pohang University of Science and Technology suggest that there have been remarkable advances to the recreation of vasculatures and large organs even though challenges remain immense.

Whereas some of the highlights of the research include novel benefits from bioprinting in space, considering that microgravity conditions enable 3D bioprinting of tissue and organ constructs of more complex geometries with voids, cavities, and tunnels; cell expansion as a critical upstream process step for cell and tissue manufacturing; the great promise of stem cells in biomedical research and applications, which through bioprinting, can be particularly positioned in 3D in relation to other cell types and/or biomaterials, as well as progress in stem cell biology and in vitro culture which is opening up new doors to regenerative medicine and better physiological cell-based assays for disease models.

With so many advances in bioprinting around the world and remaining challenges to overcome, both seasoned researchers and newcomers will find an interesting and complete summary of the bioprinting industry. This game plan can help researchers come together to create new novel processes and fill in current technological gaps, as the researchers suggest.

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3D Bioprinting Solutions: The First Bioprinting Company in Russia

3Д Биопринтинг Солюшенс, also known as 3D Bioprinting Solutions, is the only company developing b ioprinting technology and bioinks for commercial use in Russia. Back in the summer of 2014, they created the first Russian bioprinter: FABION. But there were more firsts for the company, late last year, their hardware, in the form of the OrganAut 3D bioprinter, made space history after being dispatched to the International Space Station (ISS) where Russian cosmonaut Oleg Kononenko represented his country as the first person to bioprint human cartilage tissue while in space. The Moscow-based firm is quick to become one of the big contenders of European biotechnologies, so 3DPrint.com spoke to co-founder Yusef Khesuani about how the company is preparing for the next steps in innovative biofabrication.

Scientists working at the 3D Bioprinting Solutions lab with the OrganAut

“Right now we are finishing our first set of space experiments with biofabrication of protein crystals, biofilms and cultured meat constructs onboard the ISS,” revealed Khesuani. “And by the end of this year, we plan to implement an in-situ bioprinter, which would combine a medical robotic arm and classic bioprinting extrusion technologies, allowing us to perform medical procedures and treat defects inside surgery rooms, and we plan to start with treating skin defects in mice.”

The ambitious resume is part of the company’s core purpose of advancing tissue engineering. Originally supported by the Moscow Healthcare Department and founded by INVITRO, the largest private medical lab chain in Eastern Europe, 3D Bioprinting Solutions has been delving into 3D printed organ transplants on mice for quite a while. Successfully bioprinting thyroid glands to transplant into living mice. The scientists, led by head researcher Vladimir Mironov, chose a thyroid gland as this organ is relatively simple, making it an uncomplicated subject for research work.

FABION 3D bioprinter

The bioprinting and successful transplantation of living functional mouse’s thyroid gland construct were done using FABION, a bioprinting method using nanotechnology that no longer requires the organic or artificial scaffold on which the cells are placed. But the company has since moved onto the FABION 2, a printhead, capable of bioprinting with single tissue spheroids.

“The FABION 2 bioprinter is part of the next generation, as it iterates on the same ideas [as FABION] and brings new functionality. It’s able to print complex structures using tissue spheroids and a wide range of hydrogels with different types of polymerization like thermosensitive hydrogels, multi-component hydrogels, photosensitive, Ph-sensitive and ionic-sensitive hydrogels,” explained Khesuani.

Specialist working with FABION 2 at the lab in Russia

“The key technological feature of the FABION 2 is high-speed printing with single tissue spheroids managed by a special device, developed by 3D Bioprinting Solutions. This proprietary device’s core element is a print head which operates as a “turnstile” controlling the injection and printing of single tissue spheroids. The competitive edge of this printing method is provided by the high cell density as well as the synthesis of extracellular matrix proteins within spheroids creating highly viable functional tissue constructs. Printing dispensers are calibrated using a unique laser positioning system with an unrivalled positioning accuracy of 5 μm, enabling FABION 2 to print highly complex structures,” went on the expert, who is Managing Partner of the Laboratory for Biotechnological Research of 3D Bioprinting Solutions.

The OrganAut

With FABION 2 being launched in 2016, the science and engineering team at 3D Bioprinting Solutions started focusing their efforts on a fundamentally new type of bioprinter, based on magnetic levitation in a controlled magnetic field, which would allow programmable self-assembly of tissue and organ constructs without solid scaffolds. The first prototype of this device saw the light in autumn of 2016, one year later the unique device became the OrganAut. After the first version of the printer was destroyed when the Russian spacecraft Soyuz MS-10 crashed due to a malfunction during liftoff, the company had already a second version prepped for liftoff. The second run was successful and on December 3, 2018, the bioprinter was delivered to the ISS onboard the Soyuz MS-11 manned spacecraft to perform formative biofabrication of 3D tissue and organ constructs in microgravity. For the first time in orbit, cosmonaut-researcher Kononenko printed human cartilage tissue and a rodent thyroid gland, opening up an opportunity to further expand 3D Bioprinting Solutions’ business to science (B2S) services.

Russian cosmonaut Oleg Kononenko using OrganAut in the ISS

According to Khesuani, the 3D printing community is pretty strong in Russia, particularly regarding materials for 3D printing, mainly for metal and SLA printing. With quite a few startups in the field of medical 3D printing, mainly dental, like 3DSmile. But in the realm of 3D bioprinting, they are the only company developing products in this field. Although he acknowledges that several research institutions have purchased commercially available bioprinters for their studies in the past few years.

Yusef Khesuani

Additionally, he claims that Russia has a wide range of STEM (science, technology, engineering, and mathematics) universities to collaborate with and to hire new interns and employees from. To this date, 3D Bioprinting Solutions has partnered with Moscow State University, First Moscow State Medical University, National Research Nuclear University, Moscow Institute of Physics and Technology, Stanford University, and also several undisclosed pharmaceutical and cellular agricultural companies. The company is beginning to adapt their existing technologies for cellular agriculture applications. Framework agreements have been signed with several leading startups in the field of clean meat and now they are carrying out several joint experiments using muscle cells of various species.

World Economic Forum report on STEM careers

The 3D bioprinting community in Russia could benefit from the professionals with science, technology, engineering, and mathematics degrees that are flying off the shelves since it is one of the six countries that are vigorously turning out more STEM graduates. The World Economic Forum reported that Russia comes in at fourth place, and not far behind from the United States, in producing STEM graduates, with 561,000 students. In fact, women in Russia are entering STEM fields at a higher rate than many of their Western counterparts. If this trend keeps moving upward, bioprinting companies will benefit from the continuous inflow of professionals in the field.

3D Bioprinting Solutions is a wholly own subsidiary of VIVAX BIO, a biotech company focusing on 3D bioprinting and working on a range of hardware, materials, technologies, and products that are a part of this nascent industry. Based out of New York, VIVAX BIO is more focused on sales and marketing, while their Russian counterpart is evolving through its research and development lab. Although, Khesuani revealed that in the very near future they are also planning to open a lab in New York.

The company continues working with human cells creating 3D bioprinted tissues and organoid models for drug discovery and disease modeling as a superior alternative to traditional 2D models. Along with world-renowned scientists with a lot of experience, the company hopes to use their intellectual and financial resources a strong background to coordinate successful projects that can revolutionize the world of regenerative medicine. They expect their engineering solutions to be part of the biotechnological research that will move the medical bioprinting industry forward. Much of their work weighs heavily on anticipating how their own technologies for the development of human organs from autologous stem cells will emerge within the next 25 years, and they hope to be part of the ultimate goal of their research, which is to bioprint a human kidney.

[Images: 3D Bioprinting Solutions]

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3DPOD Episode 10: 3D Printing in Space

Space exploration and the new commercial space race is a very exciting area for 3D printing. In this episode of the 3DPod Max and myself discuss the opportunities for 3D printing in space. We look at 3D printing spacecraft, 3D printing satellites, 3D printing engines, 3D printing on board space craft and 3D printing moon bases. We really enjoyed this conversation and hope that you do as well.

The first podcast on going beyond PLA is here, our interview with Direct Dimensions CEO Michael Raphael is here while our interview with design pioneer Janne is here and our interview with Scott Summit on prosthetics is here. Our episode on bioprinting is here, 3D printing in medicine is here, 3D printed guns is here. and here is the fourth industrial revolution, all of them are here. You can find them on Spotify here.

Tethers Unlimited Recycler and 3D Printer Refabricator Operational on Board the ISS

Soace manufacturing start-up Tethers Unlimited has had a tumultuous time of late. The firm which aims to develop in space manufacturing technologies and has successfully seen its Refabricator put in use on board the ISS space station now. The recycler has been installed and is now being put to use by astronaut Anne McClaine. At the same time, Tethers has had to lay off a fifth of its staff due to cash flow problems stemming from the government shutdown in the US.

Astronaut Anne McClain installing the Tethers unlimited FDM 3D printer and recycling unit on board the ISS. She appears to be wearing a rugby shirt which would be fitting since she participated in the rugby world cup as well as being a helicopter pilot with 216 missions in Iraq, engineer, a mom and an aerospace engineer.

Tethers as a firm has always been a bit of a wild ride. The company started in 1994 looking to commercialize space tethers. Tethers in space are long (tensile) cables that can be tied to satellites and other space vehicles. Long dreamt about rarely used successfully the idea is that a long tether tied to a satellite could be used for propulsion or power generation in space. An Electrodynamic tether, for example, conducts and by passing through a planet’s magnetic field. This kind of tether can use the Lorentz force (electromagnetic force) of an electrified tether against the magnetic field of a planet to push the spacecraft into a new orbit. This would save on fuel and perhaps let craft slingshot around planets more efficiently. Momentum exchange tethers may actually let the spacecraft slingshot itself into space through spinning. A bolo of a tether tied to a craft may be used to spin and propel other craft onward in their journey.

Marko Baricevic of Tethers Unlimited testing the Refabricator

Skyhooks would do the same but at much higher speeds. A space elevator is a tether tied to a craft in geosynchronous orbit above 35,000 KM in altitude which could be used to life payloads potentially inexpensively (once you build the most expensive thing ever which is also the biggest thing ever and also would need advances in material science to even be remotely feasible). Meanwhile, several 20 kilometer long tethers could together form an electric solar wind sail propelled by an electron gun shooting at these tethers to keep them in high potential while the craft spins giving the extended tethers centrifugal force and letting them stay extended enough for them to harvest force from solar wind plasma. Tethers could also be used to generate power. Tethers are amazing dream mayonnaise for making any insane space idea palatable. Tether dreams are way beyond Elon Musk’s comparatively quotidian dreams of cities of Mars and reusable rockets without Elon’s magical capital sourcing ability and media presence.

A momentum exchange tether courtesy of Tethers Unlimited

So for Tethers, the firm, going since 1994 a 3D printer and recycler onboard ISS may seem like a bit of a climb down and limited technical challenge compared to what they want to be doing. Nonetheless, for us, it is a great leap. If we conceive of astronauts spending many years in space and journeying through the solar system we know now that many unforeseen things will go wrong. Accidents will happen and valves not opening properly and nonfunctional O rings have killed astronauts. Just a few years ago a design flaw nearly caused an Italian astronaut to drown in space. If we extend our proposed space journeys to years then we know things we will not have foreseen will go wrong beyond any imaginary tolerance for failure that we can engineer away through redundancy. The perfect spacecraft may exist on the platform but it will not exist underway.

In essence, we need a magic satchel with stuff that could repair all the things in ways that we could not imagine them breaking. A combination of a 3D printer and a recycler is that magic satchel. A recycling unit can take food packaging, waste and things no longer need it and turn it into 3D printer filament which then can be printed into solutions for problems. Nonworking solutions can be recycled into iterations of better ones and all of those failures and the winner can be recycled into future solutions waiting to happen. We commonly refer to those as 3D printer filament. A spool of filament is really a seem of ideas not made yet or a roll of problems unsolved. The reason I love 3D printing and am completely obsessed with it is this idea of a recycler and 3D printer combo remaking our world forever letting us consumer while we reuse so please excuse the much more than efficient stream of words. NASA itself says that 95% of spare parts in space will never be used but they don’t know which 95% and that on the 13 tonne ISS they predict 450 Kilograms of failures each year. This in itself makes for a very compelling case for 3D printing spares.

Graphical representation of ISS logistics.

Tethers has now made an Express rack compatible recycler that is being used on board the ISS as we speak. The Refabricators objective is to,

“The Refabricator demonstrates a unique process for repeatable, closed-loop recycling plastic materials for additive manufacturing in the microgravity environment of the ISS a minimum of seven times. Samples consisting of sections of filament and standardized material testing specimens are collected from each cycle in order to quantify any degradation of material that occurs during the recycling and printing process, and enhance the understanding of the recycling process in space.”

The Refabricator

This would be quite the polymer 3D printing challenge here on earth but at least NASA is being realistic on the number of recycling cycles and material degradation of plastics which a lot of people don’t seem to know. The Refabricator is meant to show,

“Integrated recycling/3D printing capability thus provides significant cost savings by reducing the launch mass and volume required for printer feedstock while decreasing Earth reliance.”

Tethers CEO Rob Hoyt said,

“It will provide future astronauts the ability to manufacture tools, replacement parts, utensils and medical implements when they need them, and greatly reduce the logistics costs for manned space missions by reusing waste materials and minimizing the amount of replacement parts that must be launched from Earth,”

The printer was made for $2.5 million so that’s a good amount to spend on engineering a printer that works well in space and can also recycle. Tethers has additional expertise via a $10 million FabLab project to make a fab lab in space but this is separate from Made In Space‘s own 3D printer initiative. Tethers Refabricator is meant to recycle ABS and they will do it through a process that they’ve called positrusion.

As well the Positrusion effort by Tethers NASA is also developing the CRISSP both as apart of NASA’s ISP (In Space Manufacturing) program. CRISSP is focused on recycling packaging but is also being carried out by Tethers while Cornerstone Research Group is doing a similar effort (but with creating reversible copolymers that can take antistatic bags and turn them into parts) and Resonetics has been tasked with making a sensor and monitoring package. Meanwhile Made in Space is working on its printer and 3D printed metal printing for NASA. Ultratech Machinery (with ultrasonic 3D printing), Techshot and Tethers again are also working on metal parts. With Tethers opting to use its Positrusion system for metals and then combine it with a robot arm and CNC. In metals Techshot wants to use low powered lasers with metal wire in its SIMPLE technology (which is far from it). Techshot’s SIMPLE will use an induction coil around an FDM nozzle to extrude a metal filament which is then sintered by a low power laser. Techshot itself is also working on recycling and separately biofabrication. whats better than astronauts? 3D printed astronauts. Weirdly GE isn’t apparently working for NASA on metal even though its EBM process has been evaluated thoroughly by NASA. Tethers is also working on medical printing in space while the Marshall Space Flight Center itself is trying to print electronics and circuits. NASA also has efforts underway to print structures in space outside of the vehicle which Made in Space, Loral, Orbital ATK and Tethers are working on. NASA also 3D printing structures on MARS so Elon has a place to live. This MARS effort has a contest element as well as a cooperation with the US Army Corps of Engineers here on earth with the ACES initiative which we’ve covered extensively. Additionally, NASA is printing engines and more parts for space systems themselves.

Positrusion is a new filament extrusion technology that Tethers came up with specifically for space based recycling. The system can accept “miscellaneous ABS parts, it will dry and degas the input material before melting and extruding it through a die, and the cross-sectional dimensions and feed-rate of the cooling extrudate will be tightly controlled in a continuous analog of closed-die molding.”

NASA diagram of the Positrusion recycling system

In closed die molding, material is injected into a closed cold mold at high velocity while degassing removes material and creates voids that must be filled while the build material is often quickly cooled. If the Refabricator can control the gas removal and make the filament free of voids while at the same time making sure that there is no bubbling on the surface then they could have a very small form factor recycling process. Tight control of that process could give them high-quality polymer parts as well. If they could tightly collapse the system they make have a really amazing nozzle based print head that can dose and deposit accurately at one point in the future.

Dr. Allison Porter Missions Manager at Tethers Unlimited with the Refabricator

As well as ABS the system is being tested for use with Ultem 9085 this SABIC material is a UL 94-V0 rated low flame, toxicity and smoke high-performance polymer which you can here on earth get on your Stratasys system and is used widely in aerospace. For space use the Ultem would be significantly safer than ABS and a better bet going forward I should hope. Would this mean that NASA would be inclined to increase its use as build material across the space craft or in other material applications? Ultem Tang packaging anyone?

Developments as the Refabricator would seem to be absolutely essential for the future of space exploration and travel. By recycling what is on board and what is no longer used astronauts could develop solutions for many of the problems that they can encounter and extend the life of the craft that they are traveling on. Here on earth, refabricator-like devices could extend all of the things that surround us. What do you think will homes see refabricators or will this just be a tool for spacefarers? In the meantime here on Earth Tethers has just shed some very experienced people and is hoping to avoid another shutdown, a rather humdrum problem for a company that wishes to conquer the stars.