3D bioprinting tissues in space to heal people on Earth

Gravity: we notice it as the wheels of an airplane leave the ground or as we lug overflowing grocery bags up four flights of stairs. But for the most part, we don’t think about it all that often.

Cells, on the other hand, feel the effects of gravity profoundly, and they react quite strongly when it’s taken away. When researchers launched bone cells into space, they saw their cytoskeletons rearrange in odd ways, and the cells’ growth slowed to a crawl (1). Immune cells showed impaired differentiation and activation in space compared to cells that never left the ground (2).

In certain situations, however, the loss of gravity can be a good thing for cells and humanity alike. One of those cases is 3D bioprinting. 

Like typical 3D printers that expel layers of plastic on top of each other to create a structure, a 3D bioprinter uses bioink, a mixture of cells and thickening components, to print layers of cells. The main printing component of a 3D bioprinter is the printhead, which has a nozzle that releases a stream of bioink in a single layer on a surface. The printhead then goes back over that first layer of cells with another layer, following a programmed route. Scientists can take computed tomography (CT) or magnetic resonance imaging (MRI) data from a patient, use it to create a 3D model, and print a cellular structure that is completely personalized to that patient (3). To become functional tissues, these 3D printed structures need time and specialized conditions to mature.

“We are not printing tissues. We are not printing organs,” said Nieves Cubo-Mateo, a 3D printing researcher at Nebrija University. “When the cells are reorganized, when the extracellular matrix is already developed, then you can say, ‘Okay, if the tissue is developing [to do] the task it is designed to, then we can talk about functional tissues.’”

Once these cellular structures mature into tissues, scientists can use them as models for pharmacological testing or even as tissue and organ transplants one day.

When scientists print these delicate cellular layers on Earth, however, gravity pays them no favors. Researchers must use scaffolds to support the layers so that they don’t collapse into a pile of cellular goo. Because of this, it is very hard to print tissues that need intricate vasculature to function such as cardiac tissue. While many scaffolds are biocompatible or biodegradable, simply their presence in 3D printed cellular structures can affect the functionality and the shape of the final 3D printed tissue.

But in the weightlessness of space, 3D bioprints don’t need scaffolds at all.

“In the absence of gravity, you can print three dimensional structures — tissues that more closely resemble the actual structures here on Earth,” said Michael Roberts, the chief scientific officer of the International Space Station (ISS) National Laboratory. “It's a very interesting space to explore possibilities of making tissue-like products that can be used here on Earth, but they're made in space, and they have superior performance characteristics.”

Now, space agencies around the world as well as private companies are developing 3D bioprinting capabilities for use in space. They aim to create better organoid models for basic science and pharmacological testing as well as tissues and eventually organs that astronauts can print on station and either use themselves or send back down to Earth.

Learning science all over again

In a room full of watchful elementary school students, Ken Savin, the chief scientist at the space technology and infrastructure company Redwire, dropped a couple of Alka-Seltzer tablets into a glass of water. Streams of tight, tiny bubbles erupted inside the glass. As the water fizzed, Savin switched on a video of NASA astronaut Scott Kelly doing the exact same procedure aboard the ISS.

“Instead of millions of tiny bubbles, it coalesces into about three large bubbles that never come to the surface. There's no force bringing them to the surface. They just roll around,” said Savin. 

Without gravity, everything behaves differently. Air bubbles and the water surrounding them are both weightless, so the cohesive and adhesive properties of water dominate.

The problem with that is with the amount of force you need to give it to release from the nozzle, it's going to hit that surface and sort of blow up — poof! So that wasn't going to work. 
- Ken Savin, Redwire

This is just the tip of the iceberg when it comes to the new science researchers and engineers need to think about when they plan to 3D bioprint in space. Savin, who got his start in the pharmaceutical industry, got his first introduction to this new science when he joined the company Techshot, which later became a part of Redwire, to develop 3D bioprinting capability in space. When Savin joined the company, their goal was to use 3D bioprinting in microgravity to create fully functional organs to send back to Earth for transplants. 

“If you step back to what you need to do to get to finally doing organs for transplant, there's a lot of immediate steps, which are actually really beneficial to humanity,” said Savin. Some of those include using patients’ own stem cells to create personalized tissue models for drug testing or tissue transplants as well as more translatable organoids for pharmaceutical testing to move away from testing on animal models.

To get there, the Redwire team first needed to create a 3D bioprinter that worked in space. One of the first problems they had to solve was how to get the bioink off of the nozzle in the printhead. On Earth, the force to push the bioink out of the nozzle combined with the force of gravity allows the bioink to break free from the nozzle and adhere to the surface on which it's being printed. But without gravity, that process is much more difficult. Just like the Alka-Seltzer from Savin’s video, the watery bioink wants to adhere to itself much more than it wants to drop away from the nozzle on the printhead.

“It will just stick to the side of the nozzle, and then just crawl up into your device,” said Savin. To get the bioink off of the nozzle, some researchers on the team wondered whether they could shoot the bioink out of the nozzle like a syringe. 

“The problem with that is with the amount of force you need to give it to release from the nozzle, it's going to hit that surface and sort of blow up — poof! So that wasn't going to work,” Savin added. Eventually, after years of research and engineering, the Redwire team developed their updated 3D bioprinter for space called the 3D BioFabrication Facility (BFF), which flew to the ISS in November 2022.

Once the printer was aboard and installed on the ISS, the team then had to get the bioinks up to the station. Bioinks contain cells that need to stay alive and healthy, so flying them to space is not trivial.

“In that first launch when the bioinks were launched, there was an anomaly with that spacecraft, and its power system was not functioning properly. They had to turn off the refrigeration units, and that bioink failed,” said Savin. They flew more bioink up to the station, which stayed at temperature and worked well in the 3D bioprinter.

From there, astronauts on the ISS load the cartridges of bioink into the 3D bioprinter, finish the print, and then put the newly printed cellular structure into a new container that goes into a special bioreactor. They hold the bioreactor at a specific temperature, and over the course of a couple of weeks to a month and a half, they let the cellular structure grow and differentiate into their tissue of interest.

Biology is very tough to begin with. Probably the most difficult research we do on station is biological research, but it also has potentially the greatest impact on our humanity long term. 
– Ken Savin, Redwire

For instance, the Redwire team is currently collaborating with the Uniformed Services University of the Health Sciences to use the BFF to print a human meniscus on the ISS. About 60 to 70 in 100,000 people in the United States tear their knee meniscuses every year (4). It is one of the most common knee injuries.

When a tissue such as a knee meniscus is ready to head back to Earth, an astronaut can remove the cassette holding the matured tissue from the chamber, load it into a temperature-controlled box, and send it home on the next rocket back to Earth where it splashes down into the Atlantic Ocean off the coast of Florida.

“That ride back down can be a rough and tumble ride, in some cases pulling several Gs,” said Savin. “When I worked in pharma and I'd make a compound, I didn't have to worry about the vibrational G force with some compound. I'd just walk it down the hall.”

Luckily, the first 3D printed human meniscus made it back to Earth just fine. The Redwire team is now in the process of developing a stem cell bank on the ISS. When researchers want to use certain cells in a 3D bioprint, astronauts on station can proliferate and differentiate the stem cells in the bank as needed. The researchers won’t need to wait for a rocket launch to send more cells to space to do a follow up experiment. Always looking to the future, the Redwire team is developing a “next-generation” 3D bioprinter to send up to the ISS to improve on the BFF already there.

“Biology is very tough to begin with. Probably the most difficult research we do on station is biological research, but it also has potentially the greatest impact on our humanity long term,” Savin said.

A space band aid

Across the globe, the European Space Agency (ESA) also sees the potential of 3D bioprinting in space. One of their first targets is wound healing — both in space and on Earth.

“Wound healing is a bit impaired in microgravity,” said Markus Braun, the Head of the Space Life Sciences Program at the German Aerospace Center (DLR). “The idea was also to develop some kind of tool that could be used by patients who might have no access to doctors in a remote place.” 

Braun and his team had come across a study where researchers bioprinted skin cells over a wound in mice, and that skin bandage helped the wound heal much faster than in the control condition (5). They thought why not try 3D printing a cellular bandage for people using their own skin cells? To do this, Braun and his team at DLR partnered with scientists at Dresden University of Technology (TUD) and industry partners at the German space technology company OHB. 

To create the 3D printed band aid, the researchers would collect a person’s skin cells and put them in a state where they could be stable for a long period of time. Then, when it was time to make the band aid, someone could mix the person’s cells with a crosslinking agent, and print the cellular bandage directly onto that person’s wound, applying a personalized band aid. They soon found, however, that keeping cells alive and able to proliferate within the bioink was challenging. 

“Mixing two gels and then getting some kind of polymerized band aid — it's easy. But as soon as you have cells in there that need to be happy — that was really, really difficult,” said Braun. The researchers worked out the concentration of chemicals needed to support the cells in the bioink as well as figured out how best to extrude the bioink onto the skin with the optimal thickness.

After just 12 months of work, the teams created a hand-held 3D bioprinter called the Bioprint First Aid that looks a little bit like a pistol. The only thing left to do was send the Bioprint First Aid up to space. 

Once the 3D bioprinter arrived on the ISS, the researchers wanted to know if it could print a layer of bioink with the skin cells distributed equally throughout. Because they couldn’t use astronaut’s own skin cells, they swapped the cells for microparticles that are similar in size and shape to skin cells.

During his “Cosmic Kiss” mission from November 2021 to May 2022, ESA astronaut Matthias Maurer used the Bioprint First Aid to print two different bioinks made of microparticles onto a piece of foil on his leg.

“He was quite happy about the function of that thing, and he said he would be happy when he would be in space next time to have something like this,” said Braun. “It's always exciting when you see that, in the end, you have a successful story, and also the astronauts come back and tell you, ‘That was a nice experiment. It all worked perfectly, and the idea is really great.’”

Maurer then packed the print away so that it wouldn’t wrinkle or fold and sent it back down to Earth for the researchers to analyze. “We were surprised and really happy about the results,” said Braun. The microparticles had distributed evenly across the entire print, and he didn’t see any evidence of particles clustering. The TUD scientists on the team are now working on ways to improve the cellular conditions in the bioinks and evaluating the best thickening agents to improve these 3D bioprinted bandages. But they’re not just sticking with skin; they’ve also set their sights on bone.

Upside down bones

When Michael Gelinsky, a 3D printing researcher at TUD, started his laboratory, he had no plans to pursue space research. But one day in 2017, he got a call from OHB that ESA was interested in partnering with academics to investigate 3D bioprinting in space.

“Of course, we were immediately fascinated to translate that knowledge, these technologies, to application in space,” he said.

For Gelinsky, microgravity presented a clear opportunity to print and study bone tissue. Even with countermeasures such as exercise, astronauts lose bone tissue when they spend a long time in space. That bone loss looks similar to osteoporosis that people experience on Earth. 3D bioprinting to create bone made of human cells would give researchers a useful way to study osteoporosis drugs, for example.

“It's not easy to really mimic osteoporosis on Earth,” said Gelinsky. “There are some animal models in which you can partially mimic postmenopausal osteoporosis, but most are small animals like mice or rats which, in general, are not really good models for human bone, especially because the biomechanical aspects are very, very different.”

Just as this 3D bioprinting project was getting started, Cubo-Mateo joined Gelinsky’s team to collaborate on this project. She had experience with 3D bioprinting, and she already had knowledge of some of the challenges in regard to working in space.

“Before going there, I started on a proposal to create houses on the Moon with 3D printing,” she said. “There is a Moon village project, and they wanted to see how you can create a structure just with the material available in the surface of the of the Moon.”

Knowing that astronauts on the ISS would only have limited supplies to work with, Cubo-Mateo reasoned that it would be most useful for astronauts or future space settlers to source the components of bioink from biological products: the person whose cells are going into the 3D print, animal components, or even plant material. In the end, she, Gelinsky, and their colleagues landed on plasma from human blood as one of the main components of the bioink. 

“This is totally personalized because it comes with the growth factors from the patient, with the immune cells,” she said. To thicken the plasma into a functional bioink, she and her colleagues combined it with cellulose from plants and alginate from brown algae, a common food thickener. They then added self-setting calcium phosphate cement to the mixture and 3D bioprinted bone-like tissue constructs (6).

While they could successfully print bone-like tissues on Earth, they wondered how they could test their 3D bioprinting setup under microgravity conditions in a cost-effective way. There are a few ways to achieve microgravity conditions on Earth. Researchers can drop their experiments off the top of a drop tower to achieve weightlessness for a few seconds, or they can take a ride on a parabolic flight, giving them slightly longer than 25 seconds of weightlessness. But neither of those options were going to be long enough to test the 3D printing capabilities of their device. 

“I needed something that worked for hours, and in that sense, the only thing I could use was the ISS,” said Cubo-Mateo. “As you can imagine, that's super expensive for a proof of concept. So, we were wondering what can we use on Earth to simulate such conditions, again, in a low-cost fashion.”

The answer, surprisingly, came to Cubo-Mateo from somewhere close to home: “I must confess, this was an idea that came from my father,” she said. She was showing him the printer and explaining her problem, and he said, why not simply turn the printer upside down?

If the printer worked under gravity, G, and if they could also get it to work when gravity was pulling on the 3D printer components and the print in the opposite direction, -G, then it should work under the range of forces between G and -G.

“If it is stable enough to be solid, then if I don't apply any force, it will be even better,” Cubo-Mateo explained. “It was really nice when we came to the final meeting with ESA, and we presented the results. People were astonished by the simplicity of the solution.” Of course, she added, just turning the printer upside down doesn’t tell the researchers about how the cells in the bioink act under microgravity conditions, but it gave them a way to understand the stability of the material being printed. 

“This is now the standard test,” she said. “All the bioinks must be tested with this configuration to be able to go into further tests. So, it's quite simple, but so effective.”

Pushing their bioink research forward, Gelinsky and his team are now developing bioinks so that the researchers can prepare them on Earth and ship them to space in ready-to-use cartridges.

“It is not possible to just give the cartridge of bioink to the astronaut in the moment that person is stepping into the spaceship,” said Gelinsky. “There are again, complicated rules concerning safety, security, and all that stuff. And, therefore, there is a general precondition: Everything that is sent to ISS needs to be stable for at least two weeks.”

When Gelinsky and his team heard about that two-week stability requirement, they worried that the cells in their bioinks would not survive for that long without being surrounded by culture medium. But, he realized, “Nobody has checked it. Maybe we just should try it out, and then we started this big study in which at the end, half of my lab was involved.”

They found that multiple different cell types actually survived quite well when stored under standard refrigeration at 4°C for up to four weeks (7).

It was really nice when we came to the final meeting with ESA, and we presented the results. People were astonished by the simplicity of the solution.
– Nieves Cubo-Mateo, Nebrija University

“We were really astonished that not all cell types, but a number of relevant cell types, survived storage,” Gelinsky said. “When we then just brought it to room temperature, made the bioprinting, and then cultivated these bioprinted tissue constructs, then the cells recovered quite nicely.”

Moving forward, he and his team are looking for ways to optimize these bioinks for all of the different cell types they want to print. He is eager to begin testing these bioinks in space and hopes to use the 3D bioprinter that ESA plans to launch to the ISS in 2025 or 2026.

In the meantime, Gelinsky and his team had the opportunity to test their 3D bioprinting capabilities on a parabolic flight in September 2023. They used that flight to investigate the viscosity of their 3D bioprints in microgravity.

Both Gelinsky and Cubo-Mateo are excited to use what they learn about printing 3D cellular structures in space to solve health problems on Earth.

“Everything that we develop has its own applications on Earth, and if I'm able to create a bone substitute for space,” said Cubo-Mateo, “I can also use it here on Earth in areas that are isolated where there are not many resources. And in this sense, everything is translatable.”

Ordering a meniscus from space

With multiple teams gearing up their 3D bioprinting capabilities in space, a future where someone could order a custom tissue replacement from space doesn’t seem too far away. For now, the ISS is the perfect testing ground for 3D bioprinting capabilities, but the ISS won’t remain in use forever. The current plan is for the ISS to retire by 2031.

“When the International Space Station ceases operations, there’s hope of having commercial facilities in low Earth orbit that are dedicated to printing different tissues,” said Roberts. “Doing research and technology development in space, that informs how to do it better, and that has direct impacts back here on Earth.”

Before that happens, there is still plenty of 3D bioprinting research to do on the ISS. European researchers are eager to use ESA’s 3D bioprinter once it reaches the ISS in a few years, and Redwire is also pushing full steam ahead with their 3D bioprinting research goals with the BFF. After their successful 3D bioprint of a human meniscus, Redwire launched material to 3D bioprint cardiac tissue on the ISS in November 2023.

“There will come a day where things made in space will be changing people's lives on the ground on a fairly routine basis,” said Savin. “There'll be a real commercial space effort, and I will get to see it. I have no doubt.”

References

1. Hughes-Fulford, M. and Lewis, M.L. Effects of Microgravity on Osteoblast Growth Activation. Experimental Cell Research  224, 103-109 (1996).
2. Lv, H. et al. Microgravity and immune cells. J R Soc Interface  20, 20220869 (2023).
3. Otton, J.M. et al. 3D printing from cardiovascular CT: a practical guide and review. Cardiovasc Diagn Ther  7, 507-526 (2017).
4. Ahmed, I. et al. Meniscal tears are more common than previously identified, however, less than a quarter of people with a tear undergo arthroscopy. Knee Surg Sports Traumatol Arthrosc  29, 3892–3898 (2021).
5. Binder, K.W. et al. In situ bioprinting of the skin for burns. Journal of the American College of Surgeons  211, S76 (2010).
6. Ahlfeld, T. et al. A Novel Plasma-Based Bioink Stimulates Cell Proliferation and Differentiation in Bioprinted, Mineralized Constructs. ACS Applied Materials & Interfaces  12, 12557-12572 (2020).
7. Windisch, J. et al. Bioinks for Space Missions: The Influence of Long-Term Storage of Alginate-Methylcellulose-Based Bioinks on Printability as well as Cell Viability and Function. Adv Healthcare Mater  12, 2300436 (2023).