Bioprinter Ink

Vladimir Mironov: "Creating ink for 3D bioprinting is like making jelly"

Ivar Maksutov, publisher of Post-Science, talked with bioengineer Vladimir Mironov about what biochernila is, how human organs are printed and how biotechnologies will develop further.

Basics of bioprinting

– How is 3D bioprinting fundamentally different from 3D printing?

– In conventional three–dimensional printing, you can use ceramics, metal, titanium, plastic - anything. But they don't use live cells there. As soon as we start printing organs with living cells, there are a lot of restrictions: temperature, toxicity, and the like.

When our field began to develop, many believed that we simply take existing three–dimensional printers and print a scaffold on them - an auxiliary support structure. When building a house, scaffolding is used, and then the scaffolding is removed, and the house remains. The same approach is used in tissue engineering. But this cannot be called bioprinting. Bioprinting is when organs are printed with living cells at once, rather than dripping biomaterials from a pipette onto a structure printed on a printer and waiting for something to form around it. Today, this method of printing is generally accepted.

– What do you need to have to print an organ?

– Previously, to print a book, it was necessary to have paper, ink, a press, metal letters (letters) and, of course, the text of the book. To print an organ, the same thing is required, only instead of text we have a computer program, instead of paper and ink – a bio–ink, and instead of a press - a bioprinter.

A bioprinter is essentially a syringe that the robot moves in three directions and which squeezes out a gel with cells at a signal. It must have a robotic positioning system: either a Cartesian system – forward, backward, up, down, to the right – or a robotic arm, where 6-8 degrees of freedom. The computer integrates all this. The bioprinter must be in a sterile environment: either in a special office or in a laminar-box. The combination of biomaterials from which organs are created is called biochernils (English bioink).

Commercial bioprinter developed by the German company Envisiontec

– How are biochernils created and what do they look like?

– The consistency of biochernil is comparable to toothpaste, which allows you to keep the shape. After printing, the biochernils polymerize and become jelly-like. If you want the shape of the printed object to match exactly what you planned to get, you will have to use a combination that cells don't really like. It is possible to use hydrogel – biochernils, consisting of 95% water. Living cells are comfortable in hydrogel, but the printed object looks like melting ice cream. Therefore, it is necessary to find a compromise: to cause rapid polymerization of the material, but so that the cells survive. Some researchers suggest printing objects in space, others – freezing them, and others are trying to find some non-toxic chemical catalyst.

It is difficult to create a biochernil. It's like making jelly. Collagen is released from the cartilage and bones of animals during long cooking, thanks to which the broth freezes, and a jelly is obtained. Similarly, biochernils should polymerize quickly and harden after printing. Biochernils can be made from both biological and synthetic materials. Natural inks often use fibrin, a protein that is formed during blood clotting. When fibrin comes into contact with a thrombin solution, polymerization immediately occurs. Hyaluronic acid and chitosan are also used. Synthetic biochernils are made of a neutral material – polyethylene glycol, which is loaded with a variety of molecules so that it is biocompatible and convenient for printing.

Ink can even be made from decellularized organs that are extracted from pigs and human corpses. With the help of detergent Triton X-100, used in washing machines, the fat contained in the cells is dissolved. Only the woven frame remains: the capsule of the organ, the partitions of the organ and the vessels, the main component of which is collagen. The advantage of this method is that you can make ink specifically for a specific organ. But it has many disadvantages. Firstly, where to get human organs for these purposes? If you take from corpses, you need to carefully check them so that there are no diseases. Secondly, how to standardize them? Any industrial product must have guaranteed quality, and if there is a very large variability, it is difficult to predict what will happen.

You can also take cells from a patient and make them synthesize an autologous extracellular matrix, which will not cause immune rejection during transplantation. These cells can create a matrix typical of cartilage, bone, adipose tissue, and so on. And finally, it is possible to create any biomaterial, such as natural collagen, using synthetic biology methods. But Robert Langer of MIT claims that this will not happen as soon as we would like.

– And which of these methods is the most effective?

– I believe that the most promising way is to create biochernils from a person's own tissues. For example, after liposuction, the patient remains fat, collagen can be obtained from it and stored in a special tissue bank. Something happened – you take the collagen from the bank and print it. But so far this technology is very expensive.

Organs in a test tube

– A popular and favorite topic among popularizers is induced pluripotent stem cells (IPS), from which cells of any organs and tissues can be grown. In turn, IPS can be obtained from any other cells of the body by genetic reprogramming. Is it possible to combine this method with 3D bioprinting and will it give positive results?

– Japanese scientist Shinya Yamanaka received his share of the Nobel Prize for solving a very important ethical problem. The use of embryonic stem cells, which are taken from a fertilized egg that exists for only a few days, is prohibited in some countries for ethical reasons. Yamanaka learned how to obtain such cells with the help of only four genes from any cells of living adult organisms. That is, I can take a cheek biopsy, isolate a connective tissue cell, add four Yamanaki factors there, and I get an induced stem cell from which, after some time, you can make a copy of me.

Induced pluripotent stem cells are, of course, a dream. But, firstly, it is expensive, and it is not so easy to make such cells for everyone - the Japanese paid 900 thousand dollars for this experiment. Secondly, you need to change several genes at once. At the same time, to assume that nothing will happen to the genes is to know biology poorly. As soon as Yamanaka received the Nobel Prize, the Japanese strategically decided that their country should be a world leader in regenerative medicine. All procedures have been simplified, many research centers have been built. In 2014, they made a retina from induced pluripotent stem cells and transplanted it to a blind woman. She began to see. But when the retina was transplanted to the second woman, mutations arose, that is, the instability of the genome manifested itself. Clinical trials had to be discontinued.

– What organs can be made now?

– There are complex organs, there are simple ones. The simplest organ is the skin: the dermis from connective tissue and the epidermis from the epithelium. You can print cartilage: there is generally only one type of cell – chondrocytes. But with bones, everything is more complicated: vascularization is needed, that is, the formation of blood vessels. Skin and cartilage are being made now.

Swiss company Codon creates chondrospheres – groups of chondrocytes. These chondrospheres can be pipetted directly to the site of cartilage damage, without any hydrogels or scaffolds, and after two years the cartilage regenerates - it cannot be restored naturally. 15 thousand transplants have already been made, and 75% were successful. The technology is approved in the European Union. But it is complicated: firstly, mature cartilage cells are taken, which means another damage in the patient's body, and secondly, serum must be obtained from the patient's blood to grow cells outside the body. It can be made cheaper and faster and less invasive if you use not chondrocytes, but chondroblasts – differentiated stem cells that are already committed to become cartilage, but still have the ability to divide and multiply. Our research has shown that these cells can grow in any environment.

– Recently there was news that a startup Biolife4D printed a "working mini-heart". Is this PR or a real achievement?

– It's a very strange wave right now. First, Tal Dvir from Israel said that he had printed a human heart. However, the organ he printed was the size of a rabbit's heart, without valves and blood vessels. But since it looks like a human heart, he called it that. Biolife4D made a heart valve and showed that anatomically it is the same thing. In order for the heart to contract 60 times a minute for decades, powerful collagen fibers are needed. And they squeezed the resulting jelly a little and said that there were some similar mechanical properties. It seems to me that this is nonsense.

Bold prospects

– Is it really possible to grow meat artificially and how nutritious, useful and economical will it be?

– Today many companies grow meat – Memphis Meats, Gabor Forgacs. But I talked about this possibility 11 years ago. Once upon a time, PETA (People for ethical treatment of animals) came out to me. This organization fights for animal rights and protests against the killing of animals for scientific purposes. I was offered: "Here I am giving a biopsy of my muscle – isolate myoblasts from there. And we'll do an article in The New York Times that you printed a hamburger." I replied that I did not have a doctor's license and I could not take a biopsy. Besides, I don't want to engage in hi-tech cannibalism: I don't see the point of eating myself. Eventually PETA gave us a grant and we started developing the technology.

Artificial beef Mosa Meat // 3dnatives.com

Myoblasts are primitive cells from which muscle fibers are formed. To make muscle fibers from myoblasts, you need to put them next to each other and perform mechanical or electrical stimulation. Myoblasts have to grow on something - we came up with a stimuli–sensitive carrier made of chitosan, which works as a fitness center. We plant cells there, change the temperature and make them squat. All this should be in a nutrient medium in a bioreactor that controls oxygen, temperature, and the kinetics of movements. But this requires a very large grant. I wrote to all companies, organized a symposium, went to the NASA Food Center. But I was told that plants are planned to be grown on Mars, and the grant was refused.

Now everyone is talking about global warming. Animals produce a lot of greenhouse gases. Artificial cultivation of meat on an industrial scale could possibly reduce this amount. On the other hand, the process of growing meat is completely under control, which ensures sterility. I had a dream: in the evening I put frozen spheroids – 30% pork, 20% beef, and so on – in a bioreactor like a coffee machine, and by morning there is already a ready piece of meat that can be cooked in the microwave.

– Is it possible to print organs that do not exist in humans? For example, elf ears?

– There is such a possibility. The Australian artist Stelark communicated with me. He says: I want to print an ear on my cheek and talk to him. I refused, explaining to him that nerves pass there and after such an intervention the face will be distorted. In the end, he found someone who printed his ear on his hand.

You can do more serious things – for example, treat diabetes with artificially grown organs. The pancreas consists of two parts: one exocrine, which produces digestive juices, and the second endocrine is the islets of Langerhans, which produce insulin. In the Isletor project, we do not make the entire pancreas, only the endocrine part. Our doctor-collaborator took induced pluripotent stem cells, added the necessary genes there. And he had a cell that synthesizes insulin, and reacts to the concentration of glucose. He made a tissue spheroid from this cell and transplanted it under a mouse kidney capsule. In his experimental model of type 2 diabetes, when tetracycline induces diabetes, he had a normal glucose level for 5 months. I even wanted to become a volunteer.

You have read the most interesting interview in the Post-Science Control Room, and you can watch the entire broadcast here.

About the author:
Vladimir Mironov – MD, PhD, Scientific Director of the Laboratory of Biotechnological Research 3D Bioprinting Solutions.

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