No worse than "them"

In the field of regenerative medicine, the Russian Federation keeps pace with Europe and America

Serafima Chicheva, XX2 century

Regenerative medicine is in its infancy. But the dream of growing an organ "in vitro" is cherished by hundreds of scientists and doctors around the world, for the sake of it they work almost around the clock, including in Russia. One of the most important research centers in the field of regenerative medicine is located in Krasnodar. There we talked about the trends and prospects for the development of organ cultivation with the head of the Laboratory of Fundamental Research in the field of regenerative medicine, the head of the fundamental research direction of the International Research Clinical and Educational Center for Regenerative Medicine Elena Alexandrovna Gubareva.

XX2 CENTURY. At one time, many media outlets, including federal channels, spoke about the Center for Regenerative Medicine in Krasnodar. Journalists mostly stated that artificial organs are being created in Russia, but if we discard the loud headlines, what are you working on now?

E. A. Gubareva We are conducting several parallel studies aimed at studying the regeneration of intrathoracic organs and tissues. We have made the most progress in the study of regeneration of the tissues of the diaphragm and esophagus. At the beginning of the year, we managed to carry out successful operations to transplant a tissue-engineered diaphragm to rats. They replaced more than 80% of the left dome of the diaphragm with them and already on the 21st day they received an amazing result. The new tissue not only fully integrated into the environment, but also began to shrink in the absence of violations of physiological functions in animals.

Now the animals are in a chronic experiment and feel good. We observe how a fabric-engineered structure behaves. The main thing is that the diaphragm does not lose its contractile function, that is, it is not replaced by connective tissue. This is the main insidiousness of our body – any damaged tissue is replaced by connective tissue. And our goal is one hundred percent regeneration. Therefore, we are now monitoring the systemic response of the body to diaphragm transplantation.

XX2 CENTURY. At what stage are other studies?

E. G. Initially, within the framework of the Megagrant of the Government of the Russian Federation, under which we worked until December 2015, we studied the intrathoracic organs, that is, those located in the chest. At the same time, we began to create tissue-engineered matrices of the heart, lungs, diaphragm and esophagus. With organs of planar (flat) spatial organization, such as the diaphragm, everything turned out to be a little easier than with such complexly organized ones as the heart and lungs, but here we have some successes. We obtained decellularized (cell-free) matrices and learned how to sterilize the scaffolds without destroying the extracellular matrix. The next stage for us is the search for a cell line that, when seeding the frames, could give a full–fledged tissue-engineering structure.

In the case of the heart and lungs, this is a very difficult task, since these organs are complex systems consisting of a huge number of different cells, and it would be naive to believe that in 1-2 years it is possible to create a full-fledged tissue-engineered organ. Now we are talking more about creating elements of organs, the so-called patch, that is, patches. As soon as we find the most suitable cell lines, we will move further in the study of the heart and lungs.

XX2 CENTURY. What is the main difficulty in isolating cell lines? What did you use to create the aperture?

E. G. The difficulty lies in the fact that stem cells can potentially turn into malignant cells if the body's control processes over cell division and cell death are disrupted. Different cells have different degrees of risk of becoming malignant. There are proven cell lines, there are highly dangerous ones. We work with mesenchymal cells derived from bone marrow. Under normal physiological conditions, they provide regeneration. In addition, we also use mononuclear cells isolated from peripheral blood. We used these types of cells during diaphragm transplantation to rats in 2015. It is necessary to understand that it is difficult to learn how to manipulate a cell, but science is trying to learn how to select those resources that are less dangerous.

XX2 CENTURY. Tell us about the technology of creating wireframes. I understand that you are working with biological frameworks, but the whole point of regenerative medicine is to come to artificial frameworks, because this will, in fact, mean "growing an organ in a test tube".

E. G. The ability to create whole organs based even on biological frameworks will be a big breakthrough, although, of course, it will not solve the problem of donation. You see, patients who have had tissue-engineered structures transplanted, unlike patients with donor organs, do not need immunosuppressive therapy. A person who has had someone else's organ transplanted is forced to receive this therapy all his life. This significantly reduces his quality of life. Such patients often die from opportunistic infections, they should not get sick. And that's why regenerative medicine is so important, because the tissue-engineered structure does not contain foreign gene material and is not attacked by the recipient's immune cells.

But you are right, there are two options considered by regenerative medicine. This is the use of biological frameworks created on the basis of donor organs and synthetic frameworks made of polymers. We work with both, but we have achieved great success using biological frameworks.

XX2 CENTURY. Where are these frameworks created?

Decellularization of the heart

E. G. We are creating biological frameworks here in the laboratory. We take a donor organ of a laboratory animal and select a method for removing cellular material (decellularization) for it. If the organ has a developed vascular network, then we act through it by perfusion of solutions, if it is a planar organ (with a planar spatial organization), such as a diaphragm, skin or cartilage, we place it in special solutions on a rotating platform. The whole process of decellularization takes place in a special bioreactor under the influence of solutions of enzymes and detergents of various concentrations. They act as surfactants, destroy the cell membrane, it bursts, and all the contents, roughly speaking, are washed out, only the components of the extracellular matrix remain. As a result, the organ, while maintaining its three-dimensional shape, becomes completely devoid of cells and consists at this stage of collagens, elastin, fibronectin and other components of the extracellular matrix. 

Decellularization of the diaphragm

Then we evaluate the quality of decellularization, test the framework for the content of extracellular matrix components, such as collagens and elastins, and determine the amount of residual DNA. Be sure to plant these matrices on the withers of animals subcutaneously to see if they will cause an immune response, whether the matrix will germinate with vessels, whether cells will attach to it, whether they will be viable at the same time. This is a serious approach that requires a lot of time. It took almost a year and a half to work out each matrix in the case of the diaphragm. This is without taking into account the time it takes to populate the matrix with cells. In total, it takes up to 5 years to work out the methodology of one simple organ.

A rat in a device for fixing electrodes

As for synthetic frames, they are made for us by our collaborators from the Kurchatov Institute Research Center. These frames are created from various polymer materials, including natural polymers using electrospinning technology. 3D printing is also developing, but this technology in the field of regenerative medicine is only taking the first steps. Unfortunately, at the current level of technology development, it is very difficult to come up with something more successful than a biological framework. It is extremely difficult to repeat what nature has created: you face a huge number of problems…

XX2 CENTURY. Engineering or fundamental?

E. G. Fundamental. Our colleagues, physicists and engineers, say they are ready to do anything for us if we explain exactly what we need to get in the end. It is important for them to understand what diameter of the polymer fiber should be, what dimensions of the structure. But it's not just about these parameters. Biological frameworks are conceived by nature in such a way that they have a developed vascular network, and this is the basis for the germination of the framework with new vessels. Our diaphragm is all overgrown with them. And even if the cells that we have seeded are not enough to cause regeneration, then their own bone marrow stem cells will be directed along the vascular bed to this structure. And it is still impossible to create an artificially vascular network. Maybe the solution is to saturate the material with an angiogenesis activator. But even if the synthetic structure sprouts blood vessels, this does not mean that everything is in order, a chronic inflammatory process can develop in the body. Therefore, synthetic materials require careful scientific verification.

XX2 CENTURY. So you haven't transplanted artificial skeletons yet?

E. G. Colleagues from the Kurchatov Institute are now creating frameworks for us for in vitro research ("in vitro"). We populate them with cells and see what happens to them: whether they are attached to the material, whether they remain viable during prolonged cultivation, whether there is proliferation, whether the differentiation potential is preserved. These are flaps rather than organs. While we have tested several materials, they are encrypted, and the test is conducted by a double-blind method so that neither colleagues from the Kurchatov Institute nor we could influence the results. We often communicate, via Skype and in person, discuss the results of the work.

We implanted polymer frames only on the withers of rats subcutaneously to see if they cause some kind of reaction. Some do. It depends on how they are processed. And in general, the history of the behavior of cells "in vitro" and inside the body are different stories. Having good results in vitro does not mean that the cells will be just as good in the body.

XX2 CENTURY. Polymer frameworks allow the organ to grow inside the body? In general, how do wireframes behave over time?

E. G. We are just observing this with the example of the diaphragm. The rats are now 6 months old, these animals live an average of 2 years, and the most interesting thing for us will begin in a year. We will see if there will be no diaphragm tension. In principle, it should grow together with the body.

And polymer frameworks do not interfere with the growth of the organ if they are made of biodegradable materials that dissolve over time.

XX2 CENTURY. Do you think that medicine will reach the point of using exclusively polymer frameworks?

E. G. It is the dream of any doctor to open a cabinet and get a new organ for the patient. But so far it is unrealistic. Especially if we talk about the whole organ. For example, the heart must not just be created – it must be taught to beat. The conducting system of the heart is laid in the embryonic period. If we are talking about the lungs, then there are such cells – alveolocytes – that produce a surfactant – a substance that allows the alveoli not to collapse when breathing. This is also laid at the stage of embryonic development. That is, the creation of a whole functional organ is a super task. It is difficult to talk now about when we will be able to achieve this. But I really want to dream about it. There is a bit of romance in this, we all believe that it is possible, although we understand that our lives will not be enough for this.

XX2 CENTURY. You mentioned cooperation with the Kurchatov Institute. Who else do you cooperate with or conduct joint projects with?

E. G. We are open to the whole world. We cooperate with the Faculty of Fundamental Medicine of Moscow State University and hope to work together with them to create a tissue-engineered heart. We also have a collaboration with the I. M. Sechenov First Moscow State Medical University, and we are starting a project to create a tissue-engineered kidney. We are working on the technology of creating artificial leather with the Volga Federal Medical Research Center.

XX2 CENTURY. And at the international level?

E. G. We continue to cooperate with the Karolinska Institute, we hope for joint projects with colleagues from Germany.

XX2 CENTURY. That is, in the field of regenerative medicine, Russia is developing at the level of the rest of the world?

E. G. We are keeping up with the Europeans and Americans.

XX2 CENTURY. And which countries are in the lead?

E. G. Asian countries, especially China. They do not have acute ethical problems, as in the West. In China, for example, there are no strict restrictions on the use of embryonic stem cells. But no one has yet reached the clinical trials put on stream. All undergo preclinical animal studies. A promising direction for use in regenerative medicine is the creation of skin.

By the way, we plan to start a project in this direction with colleagues from Nizhny Novgorod. Skin is very much needed, everyone is waiting for this technology to treat patients in burn centers.

XX2 CENTURY. You planned to work on primate organs. Are these plans still in force?

E. G. We are working out protocols for obtaining decellularized matrices of intrathoracic organs and tissues on non-human primates in cooperation with the Institute of Primatology in Sochi. This is a huge center of federal significance, the only one in Russia. We understand the moral responsibility of working with primates and are still far from in vivo ("live") research. We get all the donor material from monkeys who died of natural causes. I think that in 1.5–2 years we will approach the transplantation of the diaphragm to primates, now we have worked out the procedure for creating a matrix and have come to the selection of cell lines.

Decellularized primate lungs

XX2 CENTURY. How many people are working in the laboratory today?

E. G. There are 10 people working in the laboratory, all of them are graduates of KubSMU. We have pathologists, surgeons, pathophysiologists in our team, there is even a dentist who is engaged in creating mathematical models based on the data obtained. There are biochemists, physicists, engineers. In addition, as I said, we are not closed from world science, we are helped by collaborations with other research institutes.

XX2 CENTURY. Have the Center's works been published in international scientific peer-reviewed journals?

E. G. We have published articles in such rating journals as Biomaterials, Mayo Clinic Proceedings, Nature Communications. We have been participating in international conferences on regenerative medicine for three years in a row, and our reports are highly appreciated by colleagues. The report on lung tissue engineering at the World Conference on Regenerative Medicine in Leipzig (Germany) last year took 1st place in the nomination "The best poster report among the works aimed at studying the regeneration of internal organs". This recognition shows that we are going in the right direction. But to do this, you need to work on yourself every day, overcome something in yourself. His laziness, his closeness, for example, to finally learn English, which opens up huge prospects for interaction with the entire scientific community. Our laboratory has high requirements for proficiency in English at a professional level.

XX2 CENTURY. How much does it cost to grow a diaphragm for a rat? Are you experiencing difficulties with financing after the end of the Megagrant?

E. G. Taking into account the fact that the creation of a tissue–engineered structure is an integrated approach, including the production of cells, matrix, long-term cultivation, etc., then for one rat the cost is about 300-400 thousand rubles. The problem is exacerbated by the fact that, unfortunately, there are no reagents necessary for operation in Russia. Many items need to be ordered abroad. By the way, in the diaphragm project we have created and patented an unparalleled, unique bioreactor for decellularization and subsequent cell seeding.

We do not experience significant problems with financing. In 2016, we started working in a different mode. On December 25, 2015, we ran out of a Megagrant for 150 million rubles, and we switched to funding from the university. This is a unique example of the support created within the framework of the Megagrant of the laboratory after its completion. We are infinitely grateful to the management and staff of KubSMU for their trust and the opportunity to continue the work that we started within the framework of the Megagrant. The university spends about 5 million rubles a year on research. Of course, if the number of projects increases, I think the amount may change. We are not limited, it is very important for us to prove every day that it is worth it, to prove it with results and publications, not to slow down.

XX2 CENTURY. What do you personally want to achieve in your work?

E. G. Regenerative medicine, with all its prospects and speed of development, requires very serious horizontal planning. I really hope that after some time we will be able to replace at least small areas of organs, for example, areas of the heart affected by myocardial infarction, lung with chronic pathology of the respiratory system or diaphragm with congenital hernias in newborns. This will already save a lot of lives and make our work not useless.

Portal "Eternal youth" http://vechnayamolodost.ru   14.11.2016