This year, the Hessian animal welfare research prize of 15,000 euros was awarded to the two scientists, Dr. Sascha Meyer dos Santos from the Institute of Clinical Pharmacology of the University of Frankfurt  and to Andreas Daus of the Technical University of Darmstadt. In addition, the science team of Prof. Wolfgang Kleinekofort and Prof. Friedemann Völklein was honored for her achievements in the development of an animal replacement technique.

Participants of the award ceremony: the Vice-President of the Hessian Parliament, Ursula Hammann (Bündnis 90/Grüne), the member of Parliament of the SPD, Dr. Pauly-Bender, the state chairman of Bündnis 90/Grüne, Kordula Schulz-Asche and the Hessian Association of the FDP, Member of Parliament, Frank Sürmann.  Hesse was the first german state to introduce such a price and awarded it for the fourth time informed the Hessian animal welfare officer, Dr. Madeleine Martin.




The laudation was held by the Hessian Minister of Environment, Lucia Puttrich.
Photo: Christiane Hohensee

In her speech  the Hessian Environment Minister Lucia Puttrich (CDU / SPD) emphazised that as an particularly important location for science, research and development Hesse wants to offer an incentive for researchers to develop further methods that can replace animal testing. The price  has to focus on „attention to a piece of research and a bit of encouragement has to be given to research in these areas." The price was shared simply because the jury recieved so many  eligible projects.


...FRAKTALKIN PLAYS A ROLE IN THE EARLY STAGES ...
Mechanism of Atherosclerosis elucidated with human-specific Tissue

The graduate biochemist Dr. Meyer dos Santos received the award for a detailed investigation of a mechanism with which the platelets stick to the insides of the blood vessels. The results are important for atherosclerosis research. Instead of genetically modified mice, the researcher has used a flow chamber system which works with human blood vessels that originate from bypass surgeries.




Award winner Dr. Meyer dos Santos received the appreciation from the Hessian Minister of the Environment, Lucia Puttrich.
Photo: Christiane Hohensee


InVitroJobs spoke to the winner.

InVitroJobs: We would like to congratulate you on your price.

Dr. Meyer dos Santos: Thank you.

InVitroJobs: What is your working group doing in general?

Dr. Meyer dos Santos: In general, we deal with atherosclerosis research and coagulation research, where our focus lies on the platelets. I focus on the adhesion of platelets to all possible matrices, ason proteins, on endothelial cells, and - what was now the core of our work - in human arteries. This allows to come close to pathological processes to detect new mechanisms, such as how atherosclerosis occurs and how the disease progresses.

InVitroJobs: There are so many existing chemokines: how did find this  particular  chemokine? What is special about Fraktalkin?

Dr. Meyer dos Santos: What makes the Fraktalkin special is that it is also  membrane-bound. Most chemokines are only soluble in blood being part of the inflammatory process. It can be located on the membrane of the endothelium, i. e. on the inner wall of the blood vessel, where it can cause the adhesion of leukocytes. This was previously known. Now we showed that it also causes adhesion of platelets outside the blood flow. That is the special feature of the Fraktalkin. I found it because it was the beginning of my thesis, to clarify the mechanism of Fraktakins.

InVitroJobs: What could be the medical significance?

Dr. Meyer dos Santos: The involvement of platelets in the inflammatory process is crucial. Ten years ago, scientists still thought that the platelets are not important in the pathogenesis process of atherosclerosis. One thought that they only come  into play when the plaque rupture occurs, i. e. in the final stage of this disease. Since  groundbreaking scientific findings  we know, that the platelets are already involved in the development of atherosclerosis. Before the leukocytes invade, the platelets are already involved and conduct the leukocytes to the site of plaque formation. They also have an arsenal of pro-inflammatory agents which they can distribute triggering within these processes. So they are somehow involved in the process from the beginning.

Our results suggest that the Fraktalkin plays an important role in the early stages of the development of atherosclerosis. That would also mean that  drugs could have an effect. The problem is that the symptoms  are not e noticeable, but perhaps only 30 or 40 years later. For this reason we cannot trace their medical significance.

InVitroJobs: Is it probably hard to tell how many animal tests you could replace with your method, isn´t it?

Dr. Meyer dos Santos: Yes, it's hard to say.

InVitroJobs: Which kind of knock-out mice are used so far?

Dr. Meyer dos Santos: Well,  in our case we would have been taken Fraktalkin-knockout mice first to show the influence of Fraktalkin on platelets. Then we had to make experiments in which we induced atherosclerosis artificially, therefore you got another ApoE knock-out mixing up the mices´ lipid metabolism pattern. Then the arteries had to be prepared in order to try to induce a thromobosis in arteries on the living animal to evaluate, in which way these processes occur. Usually, you have to apply about 10 animals per batch in such experiments. Then you have - of course – to consider an untreated control group, so at least 20 animals are used. Much more serious, however, were the use of the animals in preliminary experiments and method development. Naturally, this is always a grey area, as you never know how many animals would have been applicated to get reliable data which can be published. So, in any case one has to use far more than the 20 animals.

InVitroJobs: You have used human arteries from bypass surgeries: How long are they applicable?

Dr. Meyer dos Santos: They need to be applied rather quickly. At times we culitvated them 3 to 4 days. Other groups kept the cultures sometimes up to two weeks before they were analyzed or futher processed. Under good conditions this works quite well. The cultures stay alive for a astonishingly long time.

InVitroJobs: Does the quality of the artery pieces have an impact?

Dr. Meyer dos Santos: This is a problem, even with regard to the technical realisation. Some of them are thicker, some thinner. Some can be prepared very easily and can be exempted from the surrounding connective tissue and fat. The treatment of others in this way is almost impossible, because the process of fibrosis has already startet. Overall, we were able to prove this with a reliable test method. The biggest problem was the integration of arteries in the flow chambers which were of different thicknesses. Some preparations were too thick to be usabled. They are cut of like a roof gutter and than the blood flows over there. Above it is still a kind of cover (Ibidi sticky slides). The distance between the artery and the lid above should always be constant keeping the flow conditions in the blood flow rate constant. Fortunately we didn't have much problems with the distance. For further developments this is certainly a difficult point where we need to improve the process.

InVitroJobs: After submitting your work the reviewers said: No, you should do an in vivo testing once. You managed to avoid this. How were you able to convince the evaluators?

Dr. Meyer dos Santos: First we submitted our work in "Circulation Research". The tenor of the reviewer was: Your in vitro data are well but you need data which are less artificial and closer to the in vivo situation. Naturally, most of them proposed to use the knock-out mouse. However, one of the reviewers has suggested to take human arteries, which is the best, because you're so close to the human pathophysiology as possible. Then we had to decide: animal testing – yes or no. For me several reasons speak against the use of animals. This is partly due to my parents, if they had an annoying fly in the house, they díd not kill it, but caught it, opened the window and released it. On the other hand, some considerations were purely pragmatic, i.e. if you have something specifically human, the total proteins, the entire antibody of the flow chamber, for a mouse test you would change to change everything. Therefore, we decided fot the direct approach. After we finished the test, we submitted the manuscript to Circ Res again. They just rejected it. Then we went to Blood. They accepted the model with human arteries. I think if you're in a review process it is a matter of luck whether the reviewers accept the produced data or not.

InVitroJobs: For which future questions do you want to use your model? Do you want to evaluate and subsequently have it validated? Are you planning to put it into production?

Dr. Meyer dos Santos: I think it will be difficult to put it into production, simply because we do not have infinite arterial supply. Therefore it is very difficult to produce a salable device. In any case we are planing to use it for our further investigations. We are investigating other adhesion ligands formed by the endothelial and their capacity of platelet adhesion. And here we will definitely continue to use our system. We provide it on our technology transfer platform to other interested parties. Of course as an academic institution we are always open if people are interested in learning the technique themselves, so they come in our lab and see what we are doing. We had already requests from the pharmaceutical industry and of course we are also open to take measurements, especially when it comes to blood pharmacologically and one wants to find out whether the inflammation is affected or not. This is much easier than treating the artery preparations pharmacologically. Our aim is a standardized system in order to compensate the difference in the arteries´ wall thickness, which I mentioned earlier, so that the flow profile can be reproduced easier. This is a crucial point.

One of our plans is the following: The bypass pieces are taken from the Arteria radialis, which is the artery of the forearm, or from the Arteria marmaria, the chest wall artery. Now, we want to see how these preparations react differently when they are stimulated. There are theoretical considerations that the radial artery might be more suitable for our questions. In experiments we want to compare both artery types. The result shall be published in ALTEX (Alternatives to Animal Experiments). But this is currently only at the level of a brainstorming session. In the background we have already stored frozen preparations which we wants to use to perform immunohistochemical studies in order to see whether they can be stimulated differently, i. e. whether the protein expression patterns are different. Naturally, we want to compare their function in relation to their platelet and leukocyte adhesion in the flow chamber and we hope that we succeed in doing this next year.

InVitroJobs: Thank you very much.

Dr. Meyer dos Santos: With pleasure.



... MAKING IT ATTRACTIVE FOR INDUSTRY ...
Biosensors measure action potentials of 3D cell spheres

The engineer Andreas Daus has been honoured for the development of a biosensor based on three-dimensional cell culture systems. First of all, cardiomyocytes (heart cells) and neurons (nerve cells) were each aggregated into spherical 3D structures, so-called spheroids, using a self-designed rotator. These 0.3 millimetre spheroids were then coupled to newly developed micro-electrodes for   the analysis of cellular processes. Since both the heart and nerve cells form action potentials, this enables non-invasive measurement of alterations in electrical signal patterns. The method is suitable for testing the spheroids for pharmacologically active substances or ionising or non-ionising radiation. The innovation is the result of a collaboration between the Department of Biology at Darmstadt Technical University and the Faculty of Engineering Sciences at the Aschaffenburg University of Applied Sciences.




Award recipient Andreas Daus and the Hessian Minister for the Environment Lucia Puttrich
Photo: Christiane Hohensee


InVitroJobs spoke with the award recipient Andreas Daus.

InVitroJobs: First let me congratulate you.

Dipl. Ing. Andreas Daus: Thank you.

InVitroJobs: Can you briefly describe your findings?

Dipl. Ing. Andreas Daus: In vitro cell cultures are an attractive instrument for the replacement of animal experiments. On the one hand, for global deployment as well as for a wide acceptance, it is necessary to develop in vitro systems that act as a valid substitute models, on the other hand it is necessary to establish appropriate analytical methods which make cellular responses qualitatively and quantitatively utilisable. In my project, which is conducted in very close collaboration between the Department of Biology at the Technical University of Darmstadt and the Faculty of Engineering Sciences at the Aschaffenburg University of Applied Sciences, we were able to develop a novel biosensor system that meets these requirements. Physiologically relevant, three-dimensional cell networks were coupled to a sensor system in order to obtain functional and analytical information on physical and chemical stimuli. For example, it is possible to predict the effects of pharmacological agents. Meanwhile, the results have been published several times and presented at conferences. The Animal Welfare Research Award probably represents the ultimate appreciation.

InVitroJobs: You have developed spheroids: Is this a new idea or already an established procedure?

Dipl. Ing. Andreas Daus: I copied this method from my doctorate supervisor in Darmstadt, Prof. Paul Layer. He has been using retinal spheroids as a model for developmental studies for years. I then adapted the methodology for my cell types and coupled the spheroids to microelectrode arrays for acute electrophysiological investigations.

InVitroJobs: Heart cells consist of different cell types with different functions and channels. Do all spheres contain a sufficient number of cardiomyocyte cell types in the quantities you need?

Dipl. Ing. Andreas Daus: It is indeed important to have a heterogeneous in vitro culture available. You would not be able to generate functional spheroids from a monoculture.

InVitroJobs: Did you use differentiated cardiomyocytes and neurocytes before rotation or did you use progenitor cells?

Dipl. Ing. Andreas Daus: You can compare it with embryonic tissue. Of course there are also cells that are not completely differentiated.

InVitroJobs: What is the state of play at the moment? What do you want to do next and what plan do you have for your results in the future?

Dipl. Ing. Andreas Daus: Our goal would be to win another industrial partner who also utilises the entire system. I think we have achieved very good results on a scientific level, and this should also be brought to practical application.

InVitroJobs: Has a collaborative study also been carried out by independent groups?

Dipl. Ing. Andreas Daus: So far, two laboratories have been involved, on the one hand the group at the University in Aschaffenburg under the direction of Prof. Thielemann, on the other hand the group from the TU Darmstadt led by Prof. Layer.

InVitroJobs: Can you envisage the model entering the marketplace and being implemented in law as a prescribed in vitro method, as one possible way for testing pharmacological and toxicological substances?

Dipl.-Ing. Andreas Daus: Yes, definitely. It is not my aim to only publish scientifically and then lock myself up in my lab. I want to make my work attractive for the industry and put it into practice.

InVitroJobs: Thank you.

Dipl.-Ing. Andreas Daus: It was a pleasure, thank you.




… A LEAP FROM THE SURFACE DIRECTLY TO THE PEOPLE ...
Detection of cell viability on microchip

Prof. Wolfgang Kleinekofort, head of the physics department at the RheinMain University of Applied Sciences, and Prof. Friedemann Völklein, director of the Institute for Microtechnology (IMtech), at the RheinMain University of Applied Sciences, have been honoured for their development of a microchip that can measure the metabolic heat production of cellular networks on implant surfaces in the nanocalorimetric range. The aim is to develop an alternative method to animal tests for testing the compatibility of implants with human and animal cells, so that the use of animals can at least be partly replaced. In order to test the long-term tolerance of implants in the recipient organism, they are normally tested on animals in the pre-development stage. With the help of advanced micro-sensors which can measure the heat production of cells in the nanowatt range it is now possible to determine vitality, cell damage and rejection reactions outside an organism. A specially designed miniaturised thermal element can detect temperature fluctuations in the range of less than a ten-thousandth of a degree. When an implant is rejected, there is a rise in the temperature within the cells (fever reaction). A decrease in heat production can mean an impairment of the cells by the implant. The microchip has already been tested at the Institute for Microtechnology several times, albeit using yeast cells. Now the scientists intend to test human cardiomyocytes and keratocytes. According to the researchers, the method can also be extended to test active agents in the pharmaceutical industry.





Prof. Wolfgang Kleinekofort (left) and Prof. Friedemann Völklein (middle) with the Hessian Minister for the Environment, Lucia Puttrich.
Photo: Christiane Hohensee


InVitroJobs spoke with the two scientists after the award ceremony.

InVitroJobs: First I would like to congratulate you both on your honour. It's not a financial award, but an award nonetheless, one in honour and recognition of your wonderful work.

Prof. Kleinekofort: Money can’t motivate us any more (laughs).

Prof. Völklein: laughs.

InVitroJobs: Can you briefly outline your work?

Prof. Kleinekofort: The approach is that currently 70 per cent of all newly developed surfaces in the field of medical technology display only a moderate long-time biocompatibility. The consequences for the patient can be seen for example in the recent scandal caused by the poor quality silicone implants produced by the company PIP. Ms. Puttrich already addressed the hip prosthesis scandal. The problems are caused by inadequate surface testing for long-term tolerability. Also, in the development of surfaces there is the additional problem that many new paths are explores, only a few actually lead to the objective, that being new, usable biocompatible surfaces. Thus first surfaces are developed, which are then tested on animals and then one says, well, version A version is not so good, and we can completely forget version B, C, etc. By that time the animal experiment has already been conducted. Correspondingly you simply have to assume that 70 per cent of animal testing in the field of surface development is useless. Now our approach was more or less to leap from a surface directly to the human and saying, let’s skip the animal tests. We take the body’s own cells and apply them to the surface which is to be tested. We then observe at the cellular level how human cells behave on the surface. We hope that in the future we will be able to test the biocompatibility and cell compatibility of the surfaces on a microscopic scale.

Prof. Völklein: Yes, the technical implementation. We think that one would need a sensor that detects how human cells react to certain surfaces. One approach would be to control how the cells’ metabolism behaves when they are placed on certain surfaces. We try to thermally quantify this metabolism by detecting the cell viability with a thermal sensor which is able to detect the amount of heat caused by the cells’ metabolism, thereby determining whether the cells – spoken casually – feel well or not, whether they die or have normal metabolism, normal vitality. This in turn requires a sensor which is extremely sensitive, because the cells produce very little heat. That was the engineering challenge: to find a sensor sensitive enough to detect cell metabolic processes.

Prof. Kleinekofort: he is the only one able to do that kind of thing.

Prof. Völklein: What is important with this kind of approach is that the right people join forces, over and over again.

InVitrojobs: What kind of implants can be investigated using this approach?

Prof. Kleinekofort: absolutely any. Any surface is in direct contact with tissue and blood. Therefore it is pertinent to all implants that developed. No matter whether they are passive or active, whether it is a passive function heart valve or a silicone prosthetic, or an actively working ventricular assist device that is implanted–when it comes down to it, it’s about all surfaces that are used in medicine and cause problems.

Prof. Völklein: The flexibility results from the fact that in principle we can create all these surfaces in the sensor system using special coating processes. For example, we can create the titanium surface typically used in prostheses. You can also modify that surface in other ways, so that it always corresponds to the surface of the respective implant.

InVitrojobs: Which animals are usually used in experiments?

Prof. Kleinekofort: sheep and goats.

InVitroJobs: How do you eliminate the influence of pyrogens?

Prof. Kleinekofort: By testing for it. One can do the pyrogenicity and surface testing rolled into one. We can directly prove the fever response that is triggered by pyrogens with our chip. That is exactly what we measure in the cell. Each cell has a certain standard base performance. When it is increased, you have a fever reaction, and when it is reduced, you have toxicity. So you can see what state the cell is in by the energy directly proportional to the metabolism.

InVitroJobs: What is the current stage of development? Has the method been evaluated, or do you want to evaluate or validate it?

Prof. Völklein: The sensor works fantastically – it’s just the cells that don’t yet want do what we want.

Prof. Kleinekofort: Now we have to test many more cell lines. We have reached a point where the sensor is designed perfectly, works just fine, delivers reproducible results and we have the first cell results, but the actual qualitative and quantitative cell line research is only just beginning.

Prof. Völklein: One of the next steps will be that the working group of Prof. Thielemann in Aschaffenburg wants to place cardiac muscle cells on the chip to investigate their metabolism, so the next step will be testing the chip’s function with a very special cell system.

InVitrojobs: Are there long-term plans to bring this method to market maturity?

Prof. Kleinekofort: yes.

Prof. Völklein: yes.

InVitrojobs: You have worked with the model at the university: What kind of experiences did you have with the students? Did they find it great or too difficult?

Prof. Kleinekofort: It is difficult to find suitable people. The topic is of course appealing.

InVitrojobs: Would it be good if there were for instance a chair for replacement methods?

Prof. Kleinekofort: Yes, it would.

Prof. Völklein: Yes, it would.