Faster than on Earth

"Invitro" about the bioprinter test on the ISS

Milena Sineva, TASS

The Russian private medical company Invitro is developing a technology for printing living tissues on a 3D printer, which in the future, in particular, will allow creating full-fledged organs for transplantation. All projects are carried out by the laboratory of biotechnological research 3D Bioprinting Solutions (part of the Invitro portfolio). At the end of last year, the company sent a bioprinter "Organ.Auth" developed by scientists of the laboratory to the International Space Station. In orbit, Roscosmos cosmonaut Oleg Kononenko conducted an experiment on printing mouse thyroid tissue and human cartilage.

The project manager of the 3D Bioprinting Solutions biotechnological research laboratory, Youssef Hesuani, told TASS in an interview about the results of the experiment on board the space station, about plans to grow beef that will be useful for long-distance space flights, as well as about the possibility of printing human organs on satellites behind the Earth's magnetic field to study the effects of cosmic radiation.

– Yusef, what is a bioprinter "Organ.Auth"? What is put into it for printing organs?

– We have been developing a magnetic bioprinter "Organ.Auth" it was for the conditions of the ISS that the infrastructure already existing there was taken into account. For example, part of our experiment is carried out under certain temperature conditions, respectively, we had to put our bioprinter in a thermostat that is on the ISS. This, of course, limited the dimensions of the "Organ.Avta". Six cuvettes (removable containers where micro–organs are printed - approx. TASS) are placed inside the printer. Printing is carried out using a special material with cells. I want to note that our cuvette is more complicated than a bioprinter. The cell combines a cell delivery system to the ISS and back, as well as the system in which the experiment itself takes place. Despite the fact that it is technically very complex, the cuvette is very easy to use, there are only two buttons. One is for adding special superparamagnets immediately before the start of the printing process, the second button is for adding a special fixing agent after the formation of the micro-organ.

The American segment of the ISS has a conventional 3D printer that prints plastic, and we have a specialized apparatus for space that prints fabrics.

– What are these cages and where do you get them?

– Cells are selected depending on the scientific task. This time we printed human cartilage and mouse thyroid tissue. The cells were ordered in special "libraries" – databases that contain various cell samples, ranging from embryonic to stem cells.

– What was printed and in what quantities? How long did it take?

– We printed six samples of human cartilage tissue and mouse thyroid tissue each. On the formation of the construct (micro-organs – approx. TASS) it took a day, it's faster than on Earth. Also, the assembled organs have a better shape, the constructs are assembled in the form of an ellipse. After the formation of the tissues was completed, a fixing solution was added to them.

– Do the resulting micro-organs resemble cartilage and thyroid gland in shape?

– There are organs that must have a certain shape – aorta, uterus, bladder, trachea. And there are endocrine organs that secrete hormones, for example, the thyroid gland. With them, it absolutely does not matter what shape the printed structure will be. Moreover, you can print, for example, ten thousand small glands, but so that they work as one.

If we do not see follicles inside the construct (the thyroid gland consists of follicles – formations in the form of bubbles, the internal contents of which are a colloid – approx. TASS) thyroid glands that contain a hormone-forming colloid, then whatever external similarity we have, it will not work.

– What is the difference between the process of bioprinting in space and printing on Earth?

– In zero gravity on the ISS, the constructs are assembled perfectly in the middle of the cuvette, that is, where they should be. On Earth, because of gravity, constructs can shift to the side. In addition, during the formation of micro–organs, special substances are added to the cuvette – superparamagnets to create a magnetic field (they allow the formed construct to be held in the center of the cuvette and give it a certain shape - approx. TASS). And in space, due to weightlessness, their concentration can be reduced by an order of magnitude. This was a thing that we definitely had to check, in particular, because in high concentrations paramagnets can be toxic to cells.

– When working with "Organ.Were there any difficulties on the ISS?

– The temperature of the thermostat "walked" a little. It should remain in a certain range, since we send the cells in a gel, which turns into a liquid state at a temperature of 15 ° C and below. And the lower, the faster the rate of transition to a liquid state. The optimal rate of transition from a gel-like to a liquid state is achieved at a temperature in the range of 4-8 ° C. The thermostat kept the temperature a little higher, the gel "melted" more slowly, and I had to keep the device in the thermostat longer. The clearly prescribed timing of the experiment began to shift "to the right". It was necessary to quickly adjust the stages of the experiment online, which we did from the MCC. And I want to express my great gratitude to cosmonaut Oleg Kononenko, who conducted the experiment. He was very calm and understanding about it. As a result, according to the total time of the experiment, we managed to meet the deadline.

– What studies were carried out with the tissues after their return to Earth?

– First we had to determine that the cells inside the constructs were alive. We conducted histological studies, cut our constructs, looked at what was inside. The first result is yes, they are alive. Secondly, we saw that there were no pathological processes in the cells.

The main thing we needed to understand for the first experiment: is everything in orbit working or not, going or not going. Do the processes of self-assembly of living tissues work in the same way as on Earth? Since our technology is based on the fusion of tissue spheroids (rounded structures that consist of several thousand cells) due to surface tension forces, as well as as a result of cellular rearrangement and migration. And if it is going to, then how does all this affect the cells – whether they die or not.

After cosmonaut Sergei Prokopyev delivered the cuvettes to Earth and we received them in Moscow at Chkalovsky, we watched videos from Go-Pro cameras installed in the cuvettes in the laboratory. We looked at exactly how the process of "assembling" micro–organs was going on (the maximum length of each construct is about one centimeter - approx. TASS).

– Were the tissues collected on the ISS transplanted to mice?

— no. Because they were already in a special fixing solution, they are not functional, after that they can no longer be transplanted to mice.

Of course, if we talk about the future, we would really like to set up such experiments when we could bring micro-organs and carry out transplantation. But for this they cannot be fixed, and after printing the organs must be kept in a bioreactor. This is a system in which the nutrient fluid is pumped to maintain the viability of the construct, so that the cells grow together and interact with each other. At the first stage, our task was simply to understand that in space it is possible to print micro-organs in a bioprinter and painlessly deliver cellular material to the ISS and back to Earth. This task is completed.

– Are you considering the option for organ transplantation to be carried out on the ISS?

– The potential experiment on organ transplantation to mice on the ISS is very popular, first of all, with astronauts, because it is interesting. But this experiment in space is extremely difficult both from the point of view of the need for one of the astronauts to have surgical skills, and from the point of view of technical.

When transplanting a printed thyroid gland to mice on the Ground, it was placed under the kidney capsule (it does not have to be put in place – in the throat area), because the place under the kidney capsule is rich in blood vessels. I cannot say that this is a difficult procedure, but it is necessary to do a cavity operation: it is impossible to insert a micro-organ through micro-incisions with a syringe, there are great risks that the cells will be damaged due to the pressure created by the plunger of the syringe.

- "Organ.Avt" will be on board the ISS until 2024. Are you planning to modify the printer somehow?

– We have plans to complicate the equipment. But we will not make changes to the printer, we will make changes to the cuvette to make not a purely magnetic installation, but a magnetically acoustic one - we will superimpose acoustic waves on magnetic ones to get a more complex geometric shape.

Now, in an improved cell on Earth, we have already learned how to get rings (in orbit, the constructs were assembled in the form of an ellipse).

I see here two interesting directions for us – a vessel and a fragment of the ureter. 

– What experiments with which cells do you plan to do next on the ISS?

– We will work with different types of cells. We want to send muscle cells, for example, cow cells. Now we are working with American and Israeli startups that get a large volume of muscle cells from stem or other types of cells to create so-called artificial meat.

– Will you print minced meat?

– More like meatballs. Small meatballs of a few millimeters, maybe up to a few centimeters. In this case, we do not need to create a structure – muscle fibers, that is, print a full-fledged steak.

– What can such meat taste like?

– For the usual taste, printed tissues should contain not only meat, it should consist of three types of cells – muscle cells, connective tissue cells and adipose tissue cells. Plus, the taste is determined not only by the composition, but also by the shape.

We will definitely explore the food direction in space. In the laboratory on Earth, we already have samples of bluefin tuna, salmon and beef cells. Now I can't say that everything will definitely fly into space. First of all, we must conduct a series of experiments with these cells on Earth.

– That is, in the future, it is possible to send samples of meat and fish cells to astronauts so that they can print their own food there?

– The ideology is this: we will send them cells in very small ditches. These cells are growing very well, you can send, relatively speaking, 100 cells, and astronauts will be able to get 100 million cells from them. They will grow these cells already in space, and, accordingly, eat them. Roscosmos and NASA are very interested in these technologies.

For such self-renewing food, we will need a large amount of water to produce a large number of cells. Recirculating water can be used. The main goal is to get self–renewable food for long-distance flights to ensure autonomy from the Ground.

– Will your next experiment on the ISS have a food orientation?

– In subsequent missions, we also plan to send inanimate materials – ceramics. It can be very useful in medicine, for example, for bone replacement.

In addition, we are going to send bacteria to the ISS. Bacteria form three-dimensional structures very quickly, just like cells. And, apparently, they quickly become antibiotic-resistant due to the rapid formation of biofilm. This is apparently also due to the fact that bacteria experience serious stress in microgravity. Such experiments will be safe for astronauts, as they are carried out in completely closed systems (no liquids need to be poured) with three degrees of protection.  As a result, during long-distance space flights, new diseases may appear that we do not know about. We should try to print them in the "Organ.Avte" to the ISS and study.

– When are you planning to conduct the next experiment on the ISS?

– In August we want to send synthetic materials and, possibly, live cells.

– How many series of experiments on the space printer are planned in total?

– We have now planned more than a dozen sessions. But we'll have to split up the experiments somehow. Perhaps we will schedule two more sessions for this year, and then we will submit a request for eight more. And maybe we'll fix ten at once.

– Will you still print organs on the ISS?

– Yes, we will definitely be there. I think it would be a good idea for us to repeat the experiment with human cartilage and the thyroid gland of a mouse. Just for control: compare what we got the first time and what we get the second time. If we talk about other organs, it would be interesting for me to try to print organs from the reproductive system – for example, ovarian tissue.

– Are you planning to involve foreign colleagues in experiments on the ISS?

– Yes, we could divide the experiment into segments of the ISS within the framework of international scientific cooperation. In particular, if we talk about improving the system of delivering the received tissues to Earth for subsequent experiments with transplantation, bioreactors are needed. That is, the idea is this: we deliver materials to the Russian segment, we do the first part of the experiment there – we print micro-organs, then we transfer the cuvettes to our American colleagues so that they use bioreactors and grow these micro-organs before returning to Earth.

– At what stage do you have a contractual process with foreign colleagues?

– Now we are bringing information to our foreign colleagues about what we have achieved during the first experiment on the ISS. We tell them about our bioprinter, cuvettes, etc., so that they understand what kind of infrastructure we already have. Then we compiled several projects that we submitted to them for consideration, where it is written which operations can be made joint, based on their infrastructure. There are no official signed contracts yet.

– Earlier you said that you plan to conduct experiments with a bioprinter on microsatellites?

– Yes, on microsatellite systems. This is a very complex project, for the implementation of which, absolutely, it is necessary to create an international group. This project could unite different space agencies. The essence is to send printed micro-organs beyond the belt of the Earth's magnetic field. And there, in real time, take readings of how radiation affects certain human organs. It is better to print organs right there so that the cells are guaranteed to be alive.

The problem is urgent – ensuring human life and safety in deep space expeditions. You can send mice and rats beyond the radiation belt of the Earth to study radiation, but their regeneration systems are different. But we are well aware that this will require a huge number of completely different competencies. It is very difficult to do this work within one country.

– When, according to your estimates, will it be possible to print organs for transplantation to people?

– We are a member of the biofacturing society, and its members believe that the first printed organ will appear in the clinic no earlier than 2030. It is primarily about the replacement of skin and cartilage defects. The tissue is planned to be printed using the patient's own cells.

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