3D bioprinting
How to print a human organ?
Vladimir Mironov, MD, PhD, Scientific Director of the Laboratory of Biotechnological Research 3D Bioprinting Solutions
Post -science
Three–dimensional bioprinting is one of the promising and new directions in biomedicine. Bioprinting can be defined as a robotic layered biopublication of three-dimensional tissues and organs from living cells and biomaterials, mainly hydrogels, according to a digital model. The technology appeared two decades ago. Now there are already commercial companies that make bioprinters. There are companies that make small tissues for drug testing and toxicity assessment. There is a society engaged in biofacturing, as well as about five magazines with the name "Bioprinting". And most importantly, there are about twenty textbooks on this discipline. In principle, the area is formed.
Where did this technology come from? First of all, there is such a thing as tissue engineering: living cells are taken, support is added (we call it scaffold – this is temporary removable support from biodegradable polymers), after all this is put in a special tank, a bioreactor, and after some time a tissue is formed. Tissue engineering, although it has already existed for more than thirty years, is essentially a method based on manual production. The fundamental difference between three-dimensional bioprinting is that we use robots, usually of the Cartesian type, which move in four directions: up, down, right, left. This allows us to transfer our technology from laboratories to industrial conditions, that is, to do mass production of standard size, standard shape.
The technology of bioprinting is similar to the technology of three-dimensional printing. The fundamental difference is that you can first print construct (scaffold), and then plant the cells. And in our technology, we simultaneously print both biomaterials and living cells. That is, when we have finished the printing process, we immediately have a fabric or organ construct. Bioprinting technology is also closely related to the direction that in the 1980s was called "rapid prototyping", and then - "additive manufacturing" or "layer–by-layer production": since we are on Earth, where there is gravity, it is usually possible to print an organ only in layers (layer by layer).
There are three stages in bioprinting technology. They are very similar to the technology of layer-by-layer production, or additive manufacturing. The first stage is called "preprocessing", that is, in order to print an organ, you first need to create a digital model of it. A digital model is a virtual presentation of an organ in a computer in the form of a coordinate system. Special programs are used here. Everything is done so that you can then transfer this information about all the structures of the organ to the robot, which will read it, and then print everything. In order to get this information, three approaches are used. Or we do serial cuts and then reconstruction – like what Nikolai Pirogov did when he froze corpses and then cut them. Now it's called computed tomography. It is possible to obtain a three-dimensional image of the heart, lungs and other organs in a living person using modern methods of computed tomography. The second method: it is necessary to make serial slices on fixed samples. The third method: knowing, for example, the rules of branching of vessels, which angle of branching, what is the ratio of the parent and child segment, you can create a three-dimensional model of the vascular tree, and make a model of the entire organ around it. The result is a digital model.
The second stage is actually bioprinting, processing. We use individual cells or tissue spheroids – these are densely packed aggregates of cells, as well as biomaterial. We use hydrogels as biomaterials, because they must first be liquid so that they can be printed, and then they must immediately polymerize. At the end of the second stage – processing – we get neither organs nor tissues. We call it "fabric and organ designs". In order for them to become real organs, it is necessary to go through the third stage – postprocessing. This stage consists in the fact that there is rapid maturation, dense packing, fusion of cells and tissues and their maturation. That is, they become similar in mechanical and functional properties to fabrics.
What is the current state of the industry? Our company has five main achievements, the so-called milestones. First, we have learned how to make large–scale production of a large number of tissue spheroids, that is, cellular aggregates of standard shape and size. We have what we call building blocks. We have worked out this technology, we get a large number of spheroids, they are all standard, they are all viable, of different types, of different complexity. Secondly, we have created the first domestic three–dimensional bioprinter in Russia, which we called "Fabion": from "fab" – fabrication and "bio" - biology. This printer is among the top five printers in the world according to an independent rating. By the criterion of multifunctionality, that is, the number of functions that it can perform, we believe that it is one of the best in the world. At least, only Germans, New Zealanders and us can print spheroids. Moreover, both have not yet made printers, but are only developing, and we already have a printing system with separate spheroids.
The third element is bio–ink. It is a hydrogel that contains 95% water. It can be dispersed, create any shape, and then it polymerizes and holds this shape. We have developed pork viscous collagen as biochernils, that is, we have our own original biochernils. When we have all three of these elements, the next step is to print the organ. In 2015, for the first time in the world, we printed a functional, vascularized organ – the thyroid gland of a mouse. But it should be noted that, firstly, this is not a person, but a mouse, and secondly, we did not use stem cells, not differentiable cells, but rounded explants of the embryonic thyroid gland. That is, we took thyroid embryos and, using a special technology, isolated small balls – the rudiments of the thyroid gland – and then printed them. Unlike many other publications, we have clearly shown the function. We took radioactive iodine and completely, as in Chernobyl, killed the thyroid gland – when we did histology, there was no thyroid gland. Then we transplanted our printed organ and restored the function by 50%. The fifth, I think, is the most interesting achievement: in December 2018, we were the first in the world to send our tissue spheroids of two types into space – the rudiments of cartilage and the rudiments of the thyroid gland. In zero gravity, we have collected fabric extracts, now we are printing an article. These are the five main achievements.
Where to go next and what are the main trends? Firstly, many believe that it is possible to do so-called in situ or in vivo bioprinting. This means that printing is done directly in the operating room, and not in the laboratory. This requires other types of bioprinters, mainly articulation type, so that there are 6-7 degrees of freedom. One of the projects we are doing together with Belgians, Austrians and Californians is hair bioprinting.
Secondly, there is hybrid biofacturing, when different methods – bioprinting and others – begin to combine, so that more complex designs can be made. And thirdly, four-dimensional bioprinting. That is, you print, for example, a construction using a polymer that has memory. When you have finished printing, you either change the temperature, or direct some kind of energy source - and you have a flat piece begins to curl. This is called "four-dimensional bioprinting".
The most important achievement and success of the whole technology would be if we printed a human kidney and successfully transplanted it to a patient. Why? Because in 1954, Jeffrey Murray transplanted a natural kidney from one twin to another for the first time in the world, and in 1990 he received the Nobel Prize. Therefore, we believe that bioprinting is a very interesting direction. Firstly, it corresponds to the third or fourth industrial revolution, since we use three-dimensional printing. Secondly, it is now very fashionable to talk about the so-called digital economy, and you can't print an organ without a digital model. That is, the technology of bioprinting just includes both the third industrial revolution and the digital economy.
There is another criterion that suggests that this direction is interesting. We noticed that there is now a very great interest of young people in this technology: PhD and doctoral programs on bioprinting are already being written. There are already courses in Holland, Germany, Australia and Russia. That is, we are already starting to train bioprinters, specialists in bioprinting. If there are specialists in bioprinting, good bioprinters, good bio-ink, tissue spheroids, and if conditions are created for the transition to the clinic, then it will still be possible to print the organ sooner or later. Naturally, this will happen if there are powerful centers of collective use, centers of excellence. For example, in Singapore, America and Germany, special centers are being created, even institutes for bioprinting. We do not have such a specialized institute of bioprinting yet, but I think that sooner or later it will be created, there will be Russian specialists, there will be Russian bioprinters, Russian bio-ink. There is a chance that we will print a human organ first in the world, because we have already printed a non-human organ.
In conclusion, I would like to say that three–dimensional bioprinting is perhaps one of the most promising areas in modern medicine. Our super task is to solve once and for all the acute problem of clinical medicine – the shortage of organs for transplantation. Every day in America, for example, 22 people die because there are no kidneys for transplantation, in Europe – 18 people, in China 2 million organs are required. That is, the market is huge. As we speak, people are dying because there is no appropriate treatment. From a business point of view, this is a very good model, from a health point of view, it will save 50% of the treatment of terminal kidney diseases. From the point of view of patients, instead of going on dialysis three times a week, instead of suffering and waiting five years for a donor organ, a person receives his own kidney, printed from his cells, and continues to lead a normal life. I think this is a very interesting task, so there is a lot of enthusiasm around tissue engineering, biofacturing and especially three-dimensional printing now.
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