About bioprinting – first-hand
"We are not dependent on pigs or donors"
If the technology of organ bioprinting is put on stream,
it will look like a car assemblyNadezhda Markina, <url>
Ru" was told by the author of the first publication about 3D bioprinting technology, scientific director of the 3D Bioprinting Solutions laboratory, Professor Vladimir Mironov.
– When and who first had the idea of 3D bioprinting?
– This is not an easy question. I can just say that I was the first. But people who work in this field have their own idea of what three-dimensional bioprinting is. In my mind, this means that you print with live cells that are embedded in the printer. Before us, there were attempts to use three-dimensional printing methods to create scaffolds: polymers, ceramics, even titanium were used, but this is all without cells. A scaffold is a scaffold, a temporary support that is used to make cells stick to it. Thus, a form is created, then the cells begin to synthesize the matrix, and the scaffold degrades. I also had attempts to use it, but then we moved to a fundamentally different approach.
Firstly, we do not print scaffold, but immediately print living tissue. Secondly, we use a soft hydrogel as a framework. And the third difference is that we use the maximum possible initial concentration of cells.
If you just mix the cells with a hydrogel, they will be at a great distance from each other. But we make a tissue spheroid in which each cell contacts with fifteen neighbors, and this achieves a high density of cells, as in living tissue.
Our first article on this method was published in 2003. This is the starting point. We have introduced the term organoprinting, the term bioink, the term bioprinting. Now sometimes many people use our terminology, but try to prove that they were pioneers.
From my point of view, I have made a very important transition from scaffold to living cells. And now our point of view is becoming more and more dominant. I think enough physics, enough chemistry, we need to make biological structures.
– Now with the use of scaffolds, great successes have been achieved in regenerative medicine, for example, in Paolo Macchiarini's operation to restore the trachea. There are already practical results. How can you formulate the advantages of your technology?
– There are, of course, works by Macchiarini. Then four articles were published – on the heart (Doris Taylor), on the lung (Laura Nicholson), on the liver and, just last month, on the kidney (both recent works from Harvard University). You can also mention that Anthony Atala made a bladder with the help of scaffolds, and Lawrence Bonasar from Cornell University made an ear, but these are all flat organs, they do not have such a voluminous structure as, say, the heart, lung, liver, etc.
The criticism of their approach is very simple: if there are not enough organs for transplantation, and you use a decellurized (de-cellular) organ as a scaffold, then where will you get them? If you switch to, say, a pig, then there are problems of immunological compatibility and viral infections. And these two problems will not allow you to get permission from regulatory authorities, such as the FDA.
– In recent operations, Macchiarini uses synthetic scaffolds.
– Synthetic scaffold also has its own problems. Although it is believed that if the material degrades, if there are no oncogenic problems, then it is even better than natural. But it has been used for almost 20 years – and what is the result? Skin, cartilage, bone – and that's it, basically. There are no special breakthroughs.
– And the trachea that Macchiarini made?
– Well, the trachea is just an air duct structure. Another aspect: we use automation, robots – this is such an elegant high-tech method. If you put it on stream, create a technological line – a robot collects cells, a robot forms spheroids, a robot prints organs, and then they mature in a bioreactor – it looks like assembling a car. And, as practice shows, the higher the level of automation, robotization, the products become cheaper over time. Besides, we are all different in size. With our method, we will be able to print organs individually – by the standards of a particular person.
There is also an alternative based on induced pluripotent stem cells, which Yamanaka made, and Sergey Kiselyov is currently dealing with them. A biopsy is taken from the skin, four genes are added – the cell becomes similar to an embryonic one. This cell can be transplanted into a blastocyst, a blastocyst can be transplanted into pregnant animals – and an animal with human organs grows up. This has already been done by the Japanese on the pancreas, using a pig. Naturally, there are blood vessels, nerves, connective tissue – all from a pig. But in principle, if you add a fifth gene that does not allow the head to develop in order to get an acephalus, the surrogate mother is already legal, abortions of the acephalus fetus are also legal…
– But this is some kind of ethically very questionable way…
– It is ethically questionable when you have killed someone. In this case, this someone is not there, because the cells are from the patient, the fetus is not a person, because there is no head… But I just wanted to introduce three possible technologies: scaffolds, natural or synthetic, the use of induced pluripotent stem cells and our organoprinting technology.
From my point of view, our technology is very adaptive, very elegant, suitable for use on an industrial scale. We are not dependent on pigs or donors and are not associated with any ethical problems. And the price of the product may become low.
– Let's move on to the components of the technology itself.
– Bioprinting is not just one technology. 4-5 technologies are dominating now. You can take a liquid, photosensitive hydrogel, you can spray powder, etc. The whole bioprinting can be divided into two groups. One option is when there is a constant stream coming from a syringe or a dispenser, and the second option is when it comes in droplets. That is, either a continuous or an intermittent circuit. Most use standard existing printers and instead of polymers that are used in the industry, they take biocompatible polymers and, printing layers in different directions, make a sponge. At first it was made of polymers as hard as plastic. Then we decided to take a hydrogel instead of plastic, because you can put cells in the hydrogel. But the cell density with this method is low.
Our technology, using tissue spheroids, allows you to immediately get a high density of cells. In addition, when linear input is used, the possibilities for organizing the architecture are very limited. Although the ear can be made in this way. But we believe that if you have point structures, such as a spheroid, then, as with a mosaic, you can draw whatever you want. We have the maximum degree of freedom. From the point of view of geometry and reproduction of anatomical and histological structure, our approach is the most advanced.
– Explain what tissue spheroids are.
– A tissue spheroid is a group of cells, 15-20 thousand, that contact each other and form a three–dimensional structure. They have the shape of a ball, and this is very important from the point of view of bioprinting, because in order to print with high accuracy, the building blocks must be standardized as much as possible. This is the most convenient form.
– Is the first device in the technological line for bioprinting a robot that makes spheroids?
— no. First we have to take the cells. This is the main problem in tissue engineering: it is necessary to have a large number of cells at a certain stage of differentiation. We are trying to use cells from adipose tissue.
Firstly, if a person has excess fat removed, no one suffers about it - on the contrary. Secondly, there are already at least two machines into which you just throw a piece of fat – and after an hour, autologous human stem cells are released in large numbers. These cells can be differentiated into different lines: cartilage, bone, connective tissue – this has already been proven. Some studies say that it is possible to make ectodermal cells (skin, nerve cells) and endodermal (liver), but this is still problematic. You can, of course, get induced pluripotent cells from a skin biopsy (and the Chinese have shown that you can take cells even from urine and get induced stem cells). Robots are also used to multiply them. That is, the first stage is to get a large number of cells and, preferably, induce their differentiation in the right direction, immediately or already in spheroids – there may be different options. That is, the first machine is an automatic sorting of cells.
The second machine is a robotic biofactory of tissue spheroids. For example, a machine developed in the USA allows you to make pits in a hydrogel: if a suspension of cells is dropped into them, the cells settle and form an ideal spheroid in the pit. By changing the number of cells in the suspension and the diameter of the pits, the size of the spheroids can be controlled. But there are limitations: the larger the diameter of the spheroid, the more likely it is that the cells inside will receive less oxygen and die. There are already two companies, in the USA and in Switzerland, that do business on creating spheroids. A German company (Co.don) made spheroids that are induced in the direction of cartilage – they called them chondrospheres. These chondrospheres are already undergoing clinical testing.
– And how do cells, say, from fat, differentiate into specialized cells?
– There are two, even three options. There is differentiation that goes on by itself in the right cellular environment, but, as a rule, it does not work. An inducing factor is needed – it can be a chemical, physical or genetic factor. It is difficult to get permission for a gene factor, so now there are already companies that produce a cocktail of growth factors, these factors differentiate fat cells, say, into cartilage cells in about three weeks. We have shown that this can also be done in spheroids. That is, we turn spheroids from stem cells into organ-specific ones.
Well, when we have made a sufficient number of spheroids, the next stage is bioprinting. The bioprinter is, of course, the most important machine, but it alone is not enough. The whole line should be. And at the last stage, a bioreactor is needed for accelerated organ maturation.
– Bioprinters, as I understand it, are already on the market?
– There are four companies. Two in the USA, the most famous is Organovo (San Diego, CA, USA): they not only make printers, but create tissues and have already made a micro–liver - a small piece of liver, vascularized. It's called microtissue, and pharmaceutical companies give them half a million dollars to develop this area.
The other company only makes printers. The Swiss company (RegenHu) has already made two bioprinters – one laser, the other dispenser. They are trying to commercialize bio-ink, but in fact they call this word bio-paper, that is, hydrogel. There is also a company in Japan (CyFuse), but they have a strange idea: they are trying to make an organ, like a barbecue is made, string spheroids on rods. In total, there are companies that sell bioprinters; there are companies that say they can make organs (at the first stage for the pharmaceutical industry); and there are companies that want to use spheroids for diagnostics, for modeling diseases, etc. From this point of view, three–dimensional bioprinting is no longer a fantasy, it is real business.
Continuation of the interview with Vladimir Mironov, in which he presented the "roadmap" of his scientific group and spoke about the immediate and long-term plans, "Gazeta.Ru" will publish in the near future.Portal "Eternal youth" http://vechnayamolodost.ru