I was looking forward to seeing apoptosis in real life

The topic of cell death is reflected in all areas of Medicine, from heart attack to cancer. Jan Balvan from the Department of Pathophysiology of the Faculty of Medicine at Masaryk University focuses on what different types of cell death look like and how to differentiate between them using the holographic microscopy method in his research. He also recently published a book on cell death in collaboration with Dr. Martina Raudenská. “We wanted to make the new knowledge available for students of Medicine. It bothered me, that there is a still lot of dogma about medical students not knowing which type of cell death stimulates inflammation or that pyroptosis is a type of cell death, that occurs after infection of the cell by pathogen,” he says in the interview. This year, he received support for his current cell death research project within the TA CR GAMA 2 programme.

10 Dec 2021

RNDr. Jan Balvan, Ph.D., Faculty of Medicine, Masaryk University

How did you get into the subject of cell death?

The topic of cell death interested me even before my doctoral studies. As a part of my Master’s thesis I was already focusing on the p53 protein, which is related to this topic very closely. When I decided to begin doctoral studies, my supervisor assigned me the topic of new trends in detection of apoptosis and autophagy. At first, I was surprised, I knew there was rapid development of various methods in this area and that even though the conditions at the campus are excellent, it is not comparable to other institutions, where these ground-breaking discoveries take place and which have a long history on the subject. However, then I got lucky. I found out that the Brno University of Technology has a special holographic microscope, which can measure cell mass and is capable of fluorescent imaging. I went to see it, looking forward to seeing apoptosis in real life for the first time. There I met Aneta Křížová. In the end, the device enthused me so much that I started working with Aneta.

Your research path was not very easy, how did your research continue?

We presented the results of our research to the director of the company TESCAN, which manufactures these microscopes. He was very enthusiastic about it, because at the time, they were not sure what this microscope could be used for in biology. However, they did not want to give us the microscope at the time, so I became an employee for about three years. As application specialist I was traveling around the world, giving lectures and doing experiments, so that I could demonstrate the potential of quantitative imaging and help eliminate any bugs in the software that belongs to the microscope. I have to admit that it was very difficult task. It took me a long time to be able to set up a full-fledged experiment with results that could be published. In the end, however, it worked out.

Can you describe what the experiment consisted of?

During my travels, I’ve seen the way they deal with cell death elsewhere in the world. I had a thought that cells lose their matter during their death, which is something that can be measured with holograph. With the help of bioinformatician Tomáš Vičar we were successfully able to find other parameters typical for lytic cell death, i.e. various types of necrosis, and apoptosis, i.e. nonlytic cell death. We were able to distinguish those two processes from just the microscopic recording, without the need for any labelling of cells. This is possible using molecular methods, however, it is very indirect and sometimes misleading. We published our results. In the end we decided that we will go further with the research and try to distinguish other types of necrosis, such as pyroptosis, necroptosis and ferroptosis. These are the three types of cell death we are currently focusing on. If we were successful in distinguishing them using our method, we would be able to not only measure the dynamic parameters of a whole population over time and monitor the speed of migration or changes in cell weight, but also monitor viability and find out what percentage of cells die and how. This should have a great use in monitoring the effect of different substances, such as different nanomaterials or other experimental substances, on the monitored population.

You mentioned different types of cell death. What can I imagine hearing that?

It is a truly interesting topic, which we even wrote a book about that got published recently. For the most part, it is due to Dr. Martina Raudenská, as I am a bit messy and would not be able to work so consistently (😊). However, in the last thirty years, there have been many significant discoveries in this area. Various types of programmed necrosis were described, such as pyroptosis, necroptosis, types of cell death that cause inflammation in the body. The reach of the information from the base research to clinical practice was not as fast as we would like it to be and for that reason we wrote the book. We wanted to make new knowledge available not only to students of Medicine, but also Biology, even though Biology students do learn about cell death to some extent, so this is not a new topic for them. At the Faculty of Medicine, the information is not taught, so we wanted to promote this topic and bring it to public awareness. Cell death is reflected in all areas of Medicine from heart attack to cancer, in which case it is extremely important. It bothered me that there is still a lot of dogma between students, for example that necrosis is an unprogrammed type of cell death, that autophagy is a type of cell death, that medical students don’t know which type of cell death stimulates inflammation or that pyroptosis is a type of cell death, that occurs after infection of the cell by pathogen. Because of this we are venturing into this area and I have to say that we enjoy it so far.

You also mentioned a term that caught my attention, and that is holographic microscopy.  I can imagine what a microscope is, how it works and what I can see, but what can I see in a holographic microscope?

Basically, it is not very different from classic phase contrast, which everyone knows from the laboratory. The difference is that with a holographic microscope I am observing a hologram, which is actually a delay of light phase that is delayed only by the cell, its material, through which the light comes through a little bit slower than through a growth medium. With the use of certain formulas and calculations, we are able to calculate the mass of some hypothetic dry cell mass. Considering the fact that the background of this microscope is very well compensable, this method is the most easily accessible to various automatic algorithms for image processing. In contrast with the competition, we are, for example, able to monitor a cell or a whole population of cells for a week. And during that time, we see how their weight changed, how they died, how fast they moved.

Does that mean that a person doesn’t have to sit down and look with their own eyes?

Exactly, they don’t have to. I put a little chamber containing cells into the microscope and it records it for two days, for example. Then I can play that in a five-minute video and will get a detailed information about what happened to the cells during those two days. The main advantage though, is not the long-term recording of visual field, but the ability to quantify the recorded parameters.

Your newest project is focused on the development of a method based on holographic microscopy. Can you explain what is its principle?

The point is to capture the dynamics of cell death using different physical parameters obtained by quantitative phase imaging, including holographic microscopy. Subsequently, we subject the results to methods of deep learning, because the weight of the cells itself is not enough to distinguish different types of cell death. Apoptosis and necrosis are on the same level of cell line, which means cells of the same type, so heterogenous that I cannot distinguish them based on weight. I can find out relatively well that cells are dying, but to identify the type of death I need to involve other parameters.

Do I understand well that the result should be a programme that I could touch and if I have a holographic microscope, I would be able to watch what happens with my cell line?

If everything goes well, then yes. But these are biological systems and we have to remember that. Currently we are grappling with trying to induce ferroptosis and it is a bit of a problem, because when we pour iron there, which triggers ferroptosis, it is not enough for our cells, when we pour other specific inductors, it is still not enough and at the same time we cannot only do it on one line, which means that even if it works out for one line, it might not work for another, because it has different sensitivity to inducers.

You have completed the process of preparing an application for the support of the Proof of Concept partial project within one of the internal calls of Masaryk University, which is announced and financed by the Technology Transfer Office from the TACR GAMA 2 programme. How do you evaluate this preparation in comparison with other grants?

Considering that I have been dealing wit this issue for basically my whole career so far, I have no problems with theoretical preparation. This is not the first grant application I am submitting on this topic, which makes it easier to formulate hypotheses and goals. However, we received a lot of help from Karolína Kašparová from Office for Research and Quality FM MU. Her help was crucial, and I am not sure if I would have been able to prepare the necessary documents in time without consulting her.

Your project application has been accepted for support. As this is a grant scheme in which interaction with a commercial partner is expected, do you have any idea which companies might be interested in the results of the project?

It could be, for example, the company TELIGHT Brno s.r.o., which, unlike the parent company TESCAN, does not deal with electron microscopes, but only with light microscopy, for example super-resolution and holographic microscopes.


More about the Masaryk University project, which was supported by the TA CR GAMA 2 programme

The university-wide project within the TA CR GAMA 2 entitled “Strengthening the commercialization system of R&D results at Masaryk University” is administered by the Technology Transfer Office MU. The goal of Proof of Concept projects is to evaluate and verify the application potential of the results achieved through research and development at MU by means of so-called partial projects, which are selected for support on the basis of internal evaluation and implemented at individual MU workplaces. The award of projects is decided by the TT & Commercialization Board, which is an advisory body to the Rector and whose members are also experts from the application sphere. See more on the webpage.​

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