09 July 2013

Made in Zelenograd

Nanotechnology for the cultivation of living cells from the company NIOBIS

Elena Panasenko, Zelenograd.ruThree years ago in the studio Zelenograd.ru Roman Morozov visited – we talked about students and postgraduates of MIET, whose innovative projects were selected for funding under the Program "U.M.N.I.K." of the Fund for the Promotion of Small Forms of Enterprises in the Scientific and Technical Field (Bortnik Foundation).

Roman studied at the Department of Biomedical Systems at MIET and participated in scientific research at the Center for Probe Microscopy and Nanotechnology. "While I don't have a team, in the future I would like to create my own company," he said about his plans, as did many other hopeful young researchers.

However, the NIOBIS company was soon really created, became one of the first startups of the Zelenograd Nanocenter in 2011, and the results of its work – in the field of electrostimulation of cell growth on nanostructured surfaces – flashed at exhibitions and in the media with steady frequency. Today it is a successful project that is preparing to move to the next stage of its development and gain serious investors.
To the questions Zelenograd.ru Yuri Grishin, CEO of NIOBIS (left), and Roman Morozov, CEO of NIOBIS (right) answered.

"The prototype of the first device was assembled "on the knee", and it really worked"– The scientific basis for the creation of your company was the study of a volumetric biocompatible nanocomposite by high-resolution microscopy methods, which was carried out by a group of developers of the scientific and educational center "Probe Microscopy and Nanotechnology" (REC ZMNT) of MIET.

Is that how it all started?

Yu.G. – It all started back in 2009 – a research grant was received as part of one of the federal targeted programs, and the technology that our company is currently engaged in became a by-product of this grant. The direction of the grant was a little different, but at the same time there was such an interesting idea, and we decided to try to develop it. Then the grant funding ran out, they reported on it as expected, but the idea remained. Of course, there was no money for it, but scientists assembled a prototype of the first device "on the knee", and it really worked.

– Roman, can you tell us more about the initial research and about the "side" idea? The study was related to the creation of biocompatible implants-nanocomposites that replace cartilage tissue in animals and humans? You already talked about this in 2010...

R.M. – The initial research in which I participated took place in 2008-2009, as part of a project to create and study biocompatible nanocomposites based on carbon nanotubes, which were developed by the staff of the Department of BMS MIET. As an operator of a scanning probe microscope (SPM) at the ZMNT REC, I was engaged in determining the spatial structure of this material and tried to explain how albumin and nanotubes interact at the nanoscale level and on what principle, during laser irradiation, a material with unique properties is formed from albumin and nanotubes, different from the properties of nanotubes and albumin protein separately. This was a very non-trivial task: the samples of the material, unlike its components, were not nanoscale and were not intended for research on SPM without additional preparation, and there were no analogues of such sample preparation, however, as well as analogues of the material under study. Nevertheless, we have achieved some results: we were able to determine the spatial structure of nanocomposite samples and roughly estimate the dependence of the structure on the conditions of formation of this material.

Then there was a problem related to the certification of this material. The fact is that at that time, and still, there is no consensus in the scientific community about the toxicity of carbon nanotubes, which are a composite material of a nanocomposite. Therefore, in order to form their own opinion on this and continue work on a biocompatible composite, it was decided to investigate the interaction between nanotubes and human and animal cells for toxicity.

In an interview in 2010, I talked about this, as well as about a new development – the installation of electrostimulation of the cultivation of various cells on carbon nanotube matrices, although at that time it was only a concept in my head. The concept was confirmed after the assembly and testing of the first laboratory installation in the spring of 2011. As Yuri has already said, it was collected in a very limited time, with very modest funds.

– The issue of the toxicity of nanomaterials is still causing confusion among the people – for example, they keep writing about Petrik filters, where nanoparticles are used. What was the conclusion made at MIET then, based on the results of that study?

Yu.G. – In general, there is no definite opinion in the scientific world that nanotubes are good or bad – research is being conducted... Why, for example, did many people think they were toxic when used with cells? The problem is that they still need to be properly applied to the substrate, that's the whole point. If you arrange them randomly – I poured them out of the test tube and everything is ready – then the nanotubes do not lie down in an even layer, some are directed upwards, others somehow. And when in contact with cells, due to the fact that the nanotubes, for example, are perpendicular to the surface and the cover glass, of course, the cells are injured, the membranes burst - and the conclusion is "Well, yes, toxic."..

– This is in your device – but with natural interaction with nanoparticles, no one will smear them with an even layer when a person inhales them or receives them with water, if they are part of some solutions or, for example, in a "nanocolor"?

Yu.G. – Yes, it's better not to inhale. It is necessary to work in special rooms, there must be a hood, because after all, it is not quite good for the body.

R.M. – To be honest, I do not know what kind of particles are added to the nanocolor, whether they are there at all and whether they really have a nano-, and not a micro-size. In any case, the toxicity of nanoparticles is determined by their nature, for example, the DNA double helix has a height of 2.1 nm and is the basis for the development and functioning of living organisms and is by definition non-toxic. – The cells in your installation grow, that is, multiply by division – what exactly happens to them there?

R.M. – It is correct to say that cells are cultured. A liquid nutrient medium with cells is added to the wells (analogous to a Petri dish) of a six-hole tablet, at the bottom of which there are cover conductive glasses with a matrix of CNTs. The cells settle on the surface of the matrix, where they begin the process of division under the action of an electric field. By measuring the parameters of the field, it is possible to control the internal processes of the vital activity of cells, thus accelerating or slowing down the processes of division.

– One of the promising applications of the installation is the creation of vaccines and reducing the time for their development. How is this implemented in practice?

Yu.G. – In order to develop a new vaccine, developers need a large amount of cellular material that will be infected with the virus for further antigen isolation. Growing this biomass takes time, and we are accelerating this process. In our research, we used various cell lines, including human embryo FECH fibroblasts, green monkey cells, tumor cells (glioblastomas, Hila), nerve cells. The peculiarity of our installation is that only adhesive type cells can be grown in it, that is, cells that grow on the surface, since our technology provides for their growth on cover glasses.

– Can it be cells of internal organs, for example?

RM – Adhesive cell lines, that is, cells that divide on the surface, for example: fibroblasts, neurons, osteoblasts, stem cells. These may be heart tissue cells, skeletal muscle cells, cardiogenic cell lines.

– What are alternative cell growth technologies for the same vaccine development today?

R.M. – For example, this is the development of Ion-optix – C-Pace Culture Pacer (www.ionoptix.com ), this company has been on the market for more than five years, they have started research activities since 1994. I will clarify that from the point of view of the layman, their installation has an external similarity with ours, and its latest version, using a six-hole tablet, appeared later than our development, although it is already on sale. However, the principle of operation is different: Ion-optix acts on cultured cells with an electric field formed in a nutrient solution using a bipolar system of electrodes. Our technology is based on the effect on cells by an electric field on which they are directly cultured, that is, by the same matrix from CNT – the electric potential is supplied directly to the membrane of each cell. As a result, our technology, unlike the competitive one, allows you to reduce the applied voltage and increase the permissible exposure time, which in turn will increase the efficiency of electrical stimulation.

Another analogue is MEA Multichannelsystems from Germany, but this is a system for several other applications – such as the supply and removal of electrical signals from cultured nerve cells for the tasks of creating controlled nervous tissue.

Yu.G. – There are difficulties with analogues. The technology used there is fundamentally different, and, of course, no one gives detailed information about it – only under a non-disclosure agreement, so it's hard to say...

– Is it difficult to conduct marketing research directly in your market?

Yu.G. – Yes, of course, since this is not done in Russia, but everyone is interested in it. There are many potential customers, including various centers that work with cells, laboratories, various research institutes.

"We were invited to the Nanocenter after the "Rhythm of Zelenograd" – Who was involved in the first development of your company: scientists from MIET or students, postgraduates?

Yu.G. – Roman was engaged in the development together with two graduate students. I am an economist myself, I have been accompanying this project since 2011. So, it turned out that there was no money anymore, but there was a prototype, and we were invited to the famous annual Zelenograd exhibition "The RHYTHM of Zelenograd". Our development was noted there, and the Nanocenter was interested in it – we were offered to create a startup.

– So, you were invited to the RHYTHM, after which the startup was created? That is, does the exhibition really benefit?

Yu.G. – Yes, at first we wrote a business plan, made detailed calculations, and specialists looked at them...

– They usually complain that it takes a lot of time and effort to write all these papers and business plans - that when you go out for some kind of investment, it turns out that you are no longer doing science, but writing papers. Was there such a thing?

Yu.G. – Of course. We wrote everything for quite a long time, it was necessary to make quite detailed calculations from a technical and economic point of view, up to all taxes, etc. It is clear that this is still an estimate of a detailed business plan for activities, it will be adjusted depending on the situation, on market conditions... Of course, it took a decent amount of time, but now we have divided the responsibilities: someone is responsible for the paperwork, someone for the development.

– How much time has passed from the invitation you received at the RHYTHM to receiving the status of a startup of the Zelenograd Nanocenter?

Yu.G. – Officially, in fact, quite a lot, since we are one of the first startups of the nanocenter.

– And they also learned to work with strataps for you?

Yu.G. – Yes, and they are on us, and it was a completely new experience for us; at that time there was still no complete clarity about the requirements for a full set of documents, and in general, all this took us about a year, probably, before receiving funding. We created the company itself after the RHYTHM, in May – the RHYTHM was in the fall, which means that about six months later, when the Nanocenter had already decided that our project was really worthwhile, we need to invest, help scientists. And only then we thoroughly sat down to the business plan - when it was already clear that the game was really worth the candle.

– Is it really worth the candle, with a Nanocenter? How do you feel as one of the first startups – is it not for nothing that you got involved in this business?

Yu.G. – No, everything is fine, they really help us wherever they can, we get the opportunity to participate in exhibitions – last year we went to the Open Innovations exhibition, were participants in the collective stand of nanocenters, presented our project and the Zelenograd nanocenter. Exhibitions are an expensive pleasure, so it is difficult for a small company to go to them itself. And here they help, and yet people are already experienced, they have been working for years, so they give valuable advice. In addition, we are given access to the equipment necessary for research: microscopes, for example, which it is unrealistic for a start-up company to buy.

– Is it free for you or are you renting this equipment?

Yu.G. – There is a symbolic fee, it turns out much more profitable than renting from someone else, rather sparing conditions.

– How is this assistance being built: do you come and ask for something from the Nanocenter, or does the "boss" turn to you, constantly offer something?

Yu.G. – Bilaterally, I would say. If we have any questions, problems, they help us solve them, and in turn they offer to participate in exhibitions, for example, if there is an opportunity. Depending on the situation.

– Do you expect to receive any more grants using the help of the Nanocenter? Isn't that part of their help, too?

Yu.G. – Yes, of course. This year we are continuing development, there are certain ideas on how to bring it to the modern level – so that the interface is, as they say now, friendlier, so that it is easier to work with the installation. I think we will try to apply to the Fund for the Promotion of Small Forms of Enterprises in the Scientific and Technical Field, which is known as the Bortnik Fund, for a year to complete the entire development. Certification is needed there – our device still requires certification, and it's not fast...

– Is it possible to announce the amount of investments of ZNTC in your project?

Yu.G. – I think it is possible – 2 million rubles. Let's see how it goes from here... They can be understood: it is unwise to invest a lot of money in a company right away, you need to invest a little bit first and see how much the company really does what it needs, and not just eat up the money and that's it, "thank you, goodbye." On their part, this is also the right step to somehow secure their investments.

– Does anyone else invest in your research besides the Nanocenter?

SOUTH. – In 2012, we lived on subsidies from the Moscow department, and the Zelenograd Nanocenter invested another part of the money, 50-50 – it also invests its money, and this is just a bonus for startups.

– And MIET? Have you been affected by the law according to which a university can invest intellectual property in a startup? Or is your development not the intellectual property of MIET?

Yu.G. – No, the 217th Federal Law did not touch us. It turned out that we filed a patent together with MIET, but since there was no patent at the time of registration of the company, there could be no talk of any intellectual property.

"The creation of "artificial skin" is no longer just a prospect, but a project of a Nanocenter, for which we have become co–executors"– Now for what applications is your technology of interest in Russia?


R.M. – Both in Russia and abroad, the technology for controlling the process of cell cultivation under the influence of an electric field is of interest for such applications as: reducing the process of vaccine development – accelerating the process of cell biomass proliferation in order to isolate antigen; vaccine production – scaling technology to bioreactors; slowing down the process of tumor cell proliferation. The integration of electrical stimulation technology to accelerate the cultivation of other biological objects (for example, tea and kefir mushrooms, which are used in cosmetology) is also promising. Abroad, our experience in creating nanostructured films with specified parameters from materials whose biocompatibility is unambiguous is more interesting, and the use of such films to create three-dimensional frameworks for the cultivation of tissue cells or biocompatible matrices for tissue regeneration

– In one interview, you said that in the future your installation will be able to be used to create artificial skin. How far is this prospect?

RM – Currently, this is no longer a prospect, but a project of the ZNTC, co-executors of which are NIOBIS. We are talking about creating a matrix for the regeneration of skin structures, in fact, "artificial skin". Only it will not be used for this installation, but the technology of creating nanostructured films with specified parameters.

– Are the cells grown according to your technology not intended for transfer to the human body?

R.M – Designed. One of the possible applications of our installation is electroporation, i.e. the process of introducing nucleic acids into human and animal cells.

– Will the biocompatibility of the resulting cellular material be studied?

RM – It will be, as if there is no way without it.

– Are such cells identical to cells obtained naturally, and how can non-identity be expressed?

R.M. – No, they are not identical, since the process of introducing DNA and RNA into the cells of the body will allow changing the cellular genome and, as a result, producing genetic modification of the organism.

– What certificates and licenses are needed for your installation and technology so that they can be used in Russia, so that they can be sold? How difficult is it to get these documents? It is known about some developments of the BMS department of MIET that there were significant difficulties in obtaining Russian certificates for them – it turned out to be easier to get international ones...

R.M. – It is necessary to conduct preliminary and acceptance tests of the final experimental version of the installation, clinical trials, filing an application for state registration. When we get it, then we'll tell you if it's difficult and how much. If it will also be easier for us to get international certificates than Russian ones, we will do so. Regarding their receipt, we planned to consult with specialists from the Department of BMS.

– Do you already see the circle of potential customers who will buy the device from you when it is ready?

Yu.G. – Yes, we are working on this right now; we already have a list, and now we are working on it in detail, working out contacts.

– How many people are involved in your startup now, do you need new employees?

Yu.G. – Everything is optimal for us now, 10 people are working on the project, this is quite enough for today's range of tasks.

– And how does the Nanocenter control your work on the funds invested by it, their distribution?

Yu.G. – We provide financial statements on cash flow and a scientific and technical report once a quarter. At the very beginning, we worked out a calendar plan, outlined what tasks and in what time frame should be solved, and we are working in accordance with it.

– This calendar plan is scheduled for two years – in 2013 you will have to submit some kind of industrial design?

Yu.G. – Yes, we should already start selling. The sooner the better, of course.

– When you release an industrial design, will you leave the Nanocenter? Are there any mutual plans in this regard?

Yu.G. – In principle, several options are being considered. Now we are working on contacts with partners from Finland – they have shown interest in us, and I am in detailed correspondence with them. They wanted to invite us to their place, to see how our technology works, to conduct joint tests.

– There is a lot of biomedicine in your topic. Is there enough MIET knowledge for this? Or is there a special direction where all this is studied at MIET?

Yu.G. – The Department of Biomedical Systems is developing various equipment for use in medicine. We develop our technology with partners – this is the D.I. Ivanovsky Research Institute of Virology of the Ministry of Health of the Russian Federation, its Laboratory of Tissue Cultures, and they conduct the whole complex of biological research, and we develop the parameters of the technology itself, for example, determine what current and voltage parameters are needed, and give the result to our partners for testing. Cooperation with the Research Institute of Virology at the Department of BMS has been established for a long time, at first the guys worked with them on another project, and then everything smoothly flowed into the NIOBIS project.

R.M – The BMS Department has done and is doing its best to train technical specialists with a biomedical bias. During the first year of study, students even attend lectures and seminars at the Faculty of Biology of Moscow State University one day a week. But still, such an education is not enough to train a specialist in the field of bionanotechnology, that is, a specialist in the field located at the junction of such sciences as biology, physics, probe microscopy and nanotechnology, chemistry and medicine. At the same time, there are no universities in Russia or abroad that train such specialists, and not engineers with a biological bias or biologists with a technical bias. As, however, there are no real bionanotechnological products on the market that significantly affect the development of medicine and healthcare.

Portal "Eternal youth" http://vechnayamolodost.ru09.07.2013

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