Artificial tissues and organs may replace natural ones, however, we cannot say when it will happen, biochemist Virginija Bukelskienė, doctor of physical sciences, says in the interview. “We will have to wait and work intensively. Especially if we are talking about complex organs”, states the researcher of the Life Sciences Center of Vilnius University.
V. Bukelskienė's career has been closely linked to cells - she started her work four decades ago researching blood cancer cells, and then the world discovered stem cells, which fascinated her. It was only later, when the enthusiasm of scientists to apply these cells to therapies waned, that in-depth laboratory research began, which has been described as the return of stem cells “to the laboratory bench”.
Now the researcher’s main activity is the creation of artificial tissues and the assessment of their properties in a living organism. Dr Bukelskienė cooperates with odontologists, urologists, ophthalmologists in order to cure diseases or “correct nature’s mistakes”, and some of the methods used now would no longer be so painful.
We sat down to talk with Dr Virginija Bukelskienė about her rich career as a researcher, her favourite projects and the way they were conceived in her office at the Life Sciences Center of Vilnius University.
Q: When did you understand that you want to be a researcher?
A: I’m afraid I never understood that (laughs). I was just interested in working, and I didn’t think at all whether I’ll be a researcher… As a student, I truly liked cells: look, I thought to myself, they are taken from the body, and they grow in a test tube! How do they grow? What processes take place there? My biography is not interesting, as I have always been working in the same place, where I came when I was still a student, only the subordination of the institute changed.
Q: What was your first research? As far as I know, you were investigating stem cell possibilities.
A: Yes, I was. At the beginning, I worked with Prof. Pranas Sadauskas: we took blood of cows suffering from leucosis, cultivated its lymphocytes in vitro and evaluated the sensitivity of those cells to various substances looking for a way to stop the disease from progressing. This was both the topic of my diploma thesis and the beginning of my research work. My first supervisor was doctor Liuda Pabrėžaitė.
Then the newly started family brought about changes in my life. Upon my return to work, a semi-production, semi-research unit was just formed, in which embryonic calf serum needed for cells growth was researched and produced. It is a medium supplement rich in growth factors. In those days, buying such a serum was a huge problem, besides it was horrendously expensive. Therefore, at the beginning of the restored independent Lithuania, the production of serum was both a help to our work and a contribution to our salary. Nowadays, everything is simpler, we buy the serum from world-famous companies and it is strictly standardized, so our activity has gradually become obsolete.
As the serum production was ending, we met with cardiac surgeon Raimondas Širmenis. I don’t remember how he found us but he was greatly interested in stem cells and invited to cooperate.
I must say that this is not where my “meeting” stem cells really started. I was too young to understand then. During the production of the serum, the head of the lab Dr Vygintas Domkus already talked about stem cells. But they looked… I don’t know, kind of strange, their function in the body was unclear, so I just wasn’t interested in them. However, when R. Širmenis came with a specific proposal – perhaps to treat a human heart attack with stem cells – I “got hooked” and have been working in this field for many, many years.
Q: In the treatment of heart attack? How did you do it?
A: We chose rabbits as an experimental model as their size allowed cardiac surgeons to perform operations on the heart since, for instance, mice were too small for such a purpose. We won several projects in which we tried to apply stem cells to heart attack treatment.
We created a model in which surgeons caused a myocardial infarction to the rabbit (tying a large blood vessel in the heart) and before that, we isolated, cultured and propagated stem cells taken from the same individual’s skeletal muscles. We also carried out various genetic modifications to them to make them, how to say… more alive.
Then we would transfer those cells into the area of the infarction and see if they take, if heart function recovers etc. The rabbit would recover, live for one and a half months (this is the period allowed for it to live due to experimental conditions) then we euthanized it to assess histologically the behaviour of our cells which were stained with vital dyes, therefore, in the histological preparations we could see very beautifully and colourfully if the cells took, where they were distributed, which of them were more viable etc.
Q: Did this research develop into something more? Or it stopped?
A: Such research continued for about 10 years, and then we cooperated a lot with cardiologists, cardiac surgeons of the Vilnius University hospital Santaros klinikos. Every Friday we would go to the Santaros klinikos and draw up plans. Cardiologists were particularly interested in injecting autologous stem cells into the infarcted site. We were greatly supported by Prof. Vytautas Jonas Sirvydis. At the time, there was an idea circulating in the medical and life sciences community that chemical medicines would soon be no longer needed because all of them would be replaced by stem cells, and we believed in it.
However, research progressed, new facts appeared which dampened enthusiasm. Little by little, another phrase began to creep in stating that it was time for stem cells to return to the laboratory bench. Therefore, after dreaming that soon we will have the treatment methods, we had to come off cloud nine to carry out a lot more research to better understand those cells. In this way, the work naturally moved to the next stage, to molecular research of these cells, to another field of application of these cells.
Q: Where did you move then?
A: We made friends with physicists! With the team of Prof. Algis Petras Piskarskas of blessed memory. They began producing special frameworks with the help of lasers, and we, using stem cells, studied their toxicity or biological compatibility, evaluated how those cells grow on them and planned where such structures could be used. Gradually, the artificial tissues evolved which we grew with stem cells on the frameworks. We again carried out a lot of research and experiments, and finally, we sorted through our field: we started producing artificial bone tissue, specially preparing the frameworks they needed. We began working with odontologists, and Prof. Vygandas Rutkūnas is a great enthusiast of this research.
Then the idea of using a 3D printer to produce the frameworks was born. In the beginning, we bought such a simple, almost toy-like 3D printer. We used it to print those frameworks for tissues. Now our partners have purchased a modern 3D bioprinter, the services of which we are using.
Q: How are the artificial bone tissues you are creating applied?
A: They can be applied in a wide variety of ways. Since we cooperate with odontologists, one of the aims of their application is to cure jaw defects, as this is a very frequent problem. For example, when the tooth is missing for a long time, the bone tissue of the jaw starts dissolving, and it needs to be replenished. Currently, odontologists are using other methods as well, but the aim is to print a framework of the required personalized shape by inserting autologous stem cells into it.
Q: Currently, you are running one more project, you are developing an artificial urethra. How did you go from working with odontologists to working with urologists?
A: These works are closely related, only the physical properties of the framework are different. We continue working with odontologists, but we have also established contact with urologists.
Medical science solves a great variety of problems, and sometimes corrects nature’s mistakes as well. One of such problems is a stricture of urethra, i.e. a narrowing of the ureter. It forms due to various reasons: operations, traumas, birth defects etc. Now, a very painful surgical method is used to fix that narrowing: a patch of mucous membrane is taken from the oral cavity, with the help of which the anatomical structure of the urethra is restored.
Such operations are often performed on children. When using a patch from the mouth mucous membrane, the child has to suffer not only from the urethra but also from the mouth pain. Paediatric urologists, aware of this problem, are looking for ways to develop a better method. This is how the project Urethra was launched.
We are now working with rabbits again. We grow rabbit cells isolated from adipose tissue, embed them into a special gel, which is used to form the printed framework of the urethral tissue using a 3D printer, and then we grow the embedded cells for several days. At that stage, we seed stem cells isolated from the same rabbit’s buccal mucosa onto the surface and we get kind of a “sandwich”. The surface consists of epithelial cells from the oral mucosa, and the framework is a gel-like structure in which autologous adipose-derived stem cells from the animal are placed. This creates a patch, which is used to reconstruct the anatomical structure of the urethra.
However, the surgeons face a problem to sew it in: imagine having to sew meat jelly… but my young colleagues – Dr Egidijus Šimoliūnas and PhD student Povilas Barasa – came up with the idea of adding a thin special mesh inside the patch, which helps to solve the problem.
Q: You mentioned several times, and in your office at LSC, where we are talking now, there are plushy mice and rabbits. I’d also like to discuss the ethical side of animal research. Why are animals still used in research?
A: Lately, I’ve become an expert on this issue… In Europe over a million signatures against animal experiments have recently been collected, and quite often someone asks me to comment on the situation. In the civilized world, animal welfare is highly valued, all experiments on animals are strictly controlled, and in the European Union, it has been forbidden since 2009 to use animals to test the safety of cosmetic products. And yet, it turns out that in Britain, despite a 25-year ban there, the UK is, on the contrary, once again allowing the use of experimental animals for the testing of cosmetic ingredients in products. This is treated as a safety assessment of chemicals. I would like to say in this counterpoint that animals are still used for research in the world, and apparently may be for a long time to come, but there are very strict rules that have to be followed.
Q: Is it for accuracy?
A: Yes, because animals are like a human model. Unfortunately, our little brothers give their lives for us to gain knowledge, and the knowledge is obtained in such a painful way.
Several theories were developed around the world that underpin animal experiments. The most famous rule is the so-called 3R concept; its authors are British scientists William M.S. Russell and Rex L. Burch. In 1959, they wrote a book "Principles of Humane Experimental Techniques" on humane animal experimentation techniques. They stated that (1) there should be as few animals as possible in each experiment group; (2) animals have to be kept under ideal conditions adapted to them; and (3) alternative (non-animal) research methods should be sought whenever possible. The aim of the researchers was that animals would no longer be used at all. Such an idea existed already more than 60 years ago.
The 3R theory is reflected in documents at various levels, both in the Directive 2010/63/EU (on the protection of animals used for research purposes) and in the Order by the Director of our State Food and Veterinary Service to regulate research involving experimental animals.
Q: How is work with laboratory animals regulated? What do researchers working with animals have to do?
A: The main document we have to follow when conducting research with animals is the Directive 2010/63/EU. In Lithuania, there is a competent institution appointed by the European Commission, the State Food and Veterinary Service (SFVS), which, based on the Directive, issued the Director’s Order, which defines our entire life: how we have to carry out experiments with animals, how the laboratory has to be set up, how many staff there must be, what their qualifications are. Everything is set out very thoroughly. At the end of the document, there are forms to be filled in when submitting applications for permission to experiment on animals.
In short, each group of researchers, in order to start an experiment, fills out a long, 16–18-page application to the Ethics Commission at the SFVS, prepares summaries in Lithuanian and English (they are public and available to anyone in the world), and attaches the qualification certificates of the experimenters. Then the Commission studies the request, considers whether to grant permission.
By the way, one of the main questions in the application is determining the level of severity of the procedures, that is, whether the animal will be in pain, whether it will suffer or not, and if it suffers, what efforts the experimenter will take to make it not suffer, what drugs will use. It is a really long and responsible process, but it is highly disciplining researchers, not allowing them to work haphazardly.
Q: Listening to your answers, I see that doing your job requires a lot of creativity, curiosity and interdisciplinary collaboration – after all, you have worked with cardiologists, and physicians, and odontologists, and chemists, and urologists, and it’s not the end of the list yet?
A: Absolutely true, it is definitely needed. I’ve collaborated with a lot of people in my career, met many interesting people, and that’s how so many projects were conceived.
Q: Are there projects close to your heart personally?
A: I’ve always been interested in the borderline between in vitro and in vivo. What we study, what we change in vitro, what processes we identify and how it reflects in the body. So, you do a study in an in vitro system, you get results, but how they translate when moved into in vivo? Will the knowledge obtained in vitro be confirmed? Well, I’m interested in that borderline.
Q: You’ve implemented so many projects and research in your career, what do you spend most of your time on now?
A: To be honest, my workday is not really interesting right now. We are finishing several projects, we need to deal with various bureaucratic matters, so, going to the laboratory is very pleasant.
Q: Many researchers say the same thing: that a large share of work is taken away by bureaucracy.
A: Oh, don’t even ask… Maybe it will change in the coming generations, but now it feels like institutions that finance projects view researchers with particular suspicion.
When completing projects, all resources need to be coordinated, including working hours, staff salaries and other costs. It is at this stage that requirements sometimes lead to despair: you have to calculate four digits after the decimal point in a number and divide all this into individual activities… I understand the institutions; they make sure that the money truly goes to where it is intended. But when you need to justify everything in such a pedantic way… I don’t know…
Q: And now let’s talk about a bit more positive things, about the future. When do you think artificial tissues and organs are going to replace natural ones? Of course, the most interesting issue is to talk about such important and complex organs as the heart.
A: Maybe someday, it’ll happen, but certainly not soon. Therefore, we’ll have to wait and work intensively. In particular, if we are talking about complex organs. For example, it is far simpler with the skin, it is already being developed and even used as an alternative in cosmetic products research.
Q: In conclusion, do you notice how science is changing in Lithuania, is it, for instance, becoming more qualitative?
A: It is true, and it is for several reasons, however, the main one, no matter how banal, is increased financing – after all, reagents and equipment need money. Funding now comes in various ways: both in the implementation of Lithuania-financed projects and in working with international partners. A major turning point occurred when it became possible to buy modern equipment. There were always minds in Lithuania, but we didn’t always have things needed to work with.
Q: Thank you for your time and answers!
A: Thank you.
In the front line dr. R. Širmenis (left), Dr. V. Bukelskienė and prof. A. Piskarskas (right).
Interviewed by Goda Raibytė-Aleksa
Photo credits: V. Bukelskienė's personal archive