The annual award for the promotion of research into replacement and complementary methods for animal experiments in both scientific research and teaching is conferred by the State Office of Health and Social Affairs Berlin, together with vfa, the German Association of Research-based Pharmaceutical Companies. The award includes € 15,000 prize money, with the animal welfare organisation Bündnis Tierschutzpolitik Berlin adding € 5.000 to make a total of € 20,000.

Prof. Dr. Günter Weindl will receive the award for developing an immunocompetent skin model to replace and reduce animal tests using in vitro generated human Langerhans cells, an important immune cell type in the skin. The model is suited for testing the sensitising potential of a substance in an in vitro model. This test is currently still legally required to be conducted on mice in the so-called murine local lymph node assay following intracutaneous application of the substance to be tested.

Prof. Dr. Weindl is a pharmaceutical chemist and has been professor at the Freie Universität Berlin since 2011. In addition to developing skin models, his areas of interest include toll-like receptors, sphingosine-1-phosphate receptors and glucocorticoids. He is also the recipient of the des Heinz Maurer Award 2008 for dermatological research and the Fondation Internationale La Roche-Posay research prize.

InVitroJobs spoke with the award recipient Prof. Dr. Günther Weindl about his work and the current research situation.



Christiane Hohensee speaking with the animal welfare research award recipient,  Prof. Dr. Günther Weindl.


InVitroJobs: Professor Weindl, first of all I would like to congratulate you on receiving this award.

Prof. Weindl: Thank you very much.

InVitroJobs: What does the award mean to you, is it a great honour?

Prof. Weindl: Yes, most definitely. I feel greatly honoured by this award. When it comes down to it, I also see it as a confirmation that our research is also recognised, that many people are interested in conducting research without using animals. I know that especially from my own experience in life sciences, where a lot of work is done using mice. Numerous research groups use knock-out-models, which is why the award means a lot to me. I hope it will be an additional incentive for other scientists to become active in this area of research.




An innovative team (left to right): doctoral students Stephanie Bock, Christian Zoschke und André Said.
Image: Günther Weindl.


InVitroJobs: You have developed an immunocompetent skin model. What does “immunocompetent” actually mean, and what are the current research results that have been achieved using this model?

Prof. Weindl: Artificial skin models have been able to be grown for more than 20 years. However, the skin models consist “only” of keratinocytes¹ (see comment below) and fibroblasts². We know, however, that immune cells are also present in the skin as so-called sentinels. This means that they monitor whether substances entering the skin – such as chemicals or pathogens – represent a potential threat, and alert the immune system, which then reacts appropriately. These immunocompetent skin models now contain the Langerhans cells, which practically take over the role of sentinels.

Doctoral student Lisa Grohmann presenting a complete skin model used for pharmacological tests.


InVitroJobs: What exactly do the immune sentinels do?

Prof. Weindl: The cells are capable of discerning whether the intruding microorganism is potentially dangerous or not. For instance, there are very many bacteria in the natural skin environment. The Langerhans cells don’t react to these under normal conditions, otherwise one would always have an inflammation. On the other hand – we know this from our experiments and it is also well-known in research – contact with dangerous pathogens such as viruses activates these cells. The cells then produce a messenger substance that migrates to the lymph nodes, alerting T cells³ and other important immune cells in order to stave off the intruding pathogens.

InVitroJobs: ...and the Langerhans cells can recognise these with their surface receptors⁴?

Prof. Weindl: Exactly. There are surface receptors on the cells. If I can use the example of the pathogen again, nowadays we know of a certain receptor family, the so-called toll-like receptors, that is able to recognise preserved structures of pathogens. A well-known example is the lipopolysaccharides, part of the bacterial cell wall. The receptors are situated on the surface of the immune cells, the structures are recognised and a further reaction is either triggered or not.



Issue of honour: immunocompetent skin model.
Image: Günther Weindl.


InVitroJobs: There is a whole factory in Stuttgart that produces 6,000 skin particles. Are these to be completely replaced by your immunocompetent models later on?

Prof. Weindl: That would be a medium-term goal, if the production can be automated, but at present cultivating immune cells is still relatively difficult. We too had to learn that in our own lab. We started from scratch, concentrated on single immune cells, investigated these more closely and have now integrated them in skin models. We can’t yet automate the production, but I think we can consider that within the next, one to three years – assuming we can use the models to emulate the reactions as we see them in humans.

InVitroJobs: How do you verify the concurrence of immune reactions in vitro and in vivo?

Prof. Weindl: We rely on investigations conducted on humans who were accidentally exposed to chemicals and experienced certain skin reactions. We take those chemicals and investigate whether we reach a result similar to the one observed in the human.

InVitroJobs: You said that animals were originally used for investigating such questions. What animals were used, and how many?

Prof. Weindl: In the area of skin sensitisation, in which we are operating, mostly guinea pigs are used, mice to a limited extent – for instance in the local lymph node assay5, which is still used by the OECD as the gold standard for investigating skin sensitisation. It is hard to determine exactly how many animal tests are avoided. There are statistics that state how many animals are used per year for investigating these substances, and that’s as much as 10,000 in Germany. The number of mice used in basic research is much higher, more than a million, and here too we want our model to make an important contribution to reducing animal experiments.

At present I don’t see our model as a complete replacement for all experiments, that would be presumptuous. I do think that if we can prove the functionality of our models, we could indeed save as many as ten thousand animals.

InVitroJobs: Mice and guinea pigs.

Prof. Weindl: Tests using mice and guinea pigs are currently required by the OECD and are therefore implemented. The ECVAM⁶, which is responsible for assessing new alternatives to animal tests, issued a strategy for testing sensitising substances in March of this year⁷. This strategy encompasses the so-called Integrated Testing Strategy (ITS)⁸ and we see the skin model as a possible addition to this strategy. Fundamental research is also very close to our hearts.

We want to serve two fields, not only testing chemicals and sensitising substances, but also replacing animal experiments in fundamental research that for instance pertain to inflammatory skin diseases, psoriasis, neurodermitis. We also want to conduct tests for new potential pharmaceutical drugs with these skin models.

InVitroJobs: Until now, Langerhans cells were considered to be hard to grow. Why was it so hard?

Prof. Weindl: We are not the first to try something like that. There are already publications in which similar skin models have been created, with varying success. My personal experience tells me that sometimes it works well, sometimes not so well. In part it’s a game of chance; whether the immune cells stay in the model depends on many factors. It’s an artificial system, and for instance additives can influence the cells. We have tried using different media, altered the growing conditions, and we haven’t yet reached the end of the line. We are continuing to optimise our model, so that we can obtain genuinely reproducible results. In the end, it is the sustained work that is necessary, and testing – and then making such skin models seems possible to us.


Langerhans cells can be cultivated. Like other cells in the innate immune system, Langerhans cells can be cultivated from precursor cells isolated from human donor blood samples. To this end, scientists use a modern technique, MACS (magnetic-activated cell sorting). With the aid of antibody binding, the cells with the desired specific differentiation marker (a protein) on their surface can be sorted out.




After sorting out the desired cells, these can be stimulated to differentiate using certain cytokines.
In vitro generation of immature Langerhans cells from precursor cells in the blood (monocytes).
Illustration: André Said.

InVitroJobs: Does that mean that it’s still too early for a prevalidation study?

Prof. Weindl: Yes, it is still too early for that. We are now at the next stage; the week after next we will treat the skin models with known sensitising substances and investigate whether our cells also migrate from the epidermis⁹, so as to deliver an initial functional verification. We will then test that with further substances. Only when we have completed these tests can we consider moving on to prevalidation and test on a large scale. At the moment it’s still too early for that.

InVitroJobs: But you already aim to have the model adopted as an approved alternative to animal testing methods in the OECD test guidelines.

Prof. Weindl: We would of course wish that to happen. Right now I can’t yet judge the prospects. I know that very many test systems, including some simpler models have been submitted to EVCAM and are being examined. I personally can’t form an opinion as to whether such a skin model can also become part of a test guideline. But of course such as success would certainly be very nice.

InVitroJobs: Have industrial companies shown any interest?

Prof. Weindl: We don’t yet have any actual contacts. The industry is reticent with regard to alternative methods, with some exceptions. They have guinea pigs and mice in their labs, and it’s business as usual. The model that we grow is involves a lot of work and you need a lot of know-how to prepare the immune cells to have the right condition. That goes beyond routine work with cell cultures, so our medium-term goal is to develop a protocol that is as simple as possible, allowing a model to be used as widely as possible. At the moment I think it’s still too costly and time-consuming to produce such a model for industrial use.

InVitroJobs: How interested are students and young scientists?

Prof. Weindl: I know from pharmaceutical chemistry and my discussions with students that many students are interested in animal-free methods. Professor Schäfer-Korting’s entire department is working on animal-free methods, and we are creating different skin models. We see considerable interest on the side of the students, even if you do see pharmaceutical chemistry and especially pharmacology as a classical discipline in which animal tests are conducted.

The students are very interested and many expressly want the subjects of their doctoral theses to be ones in which no animal tests are conducted.

Therefore it makes me even happier that the award allows us to continue our work. Making these skin models is not very cheap. Chemicals, reagents and especially cytokines are very costly. It is wonderful for us that this prize money is now available for further research.

InVitroJobs: Thank you for the interview.

Prof. Weindl: It’s been a pleasure.


Literature:

Bätz, F. M., Klipper, W., Korting, H. C., Henkler, F., Landsiedel,, Luch, A., von Fritschen, U., Weindl, G. & Schäfer-Korting, M. (2013): Esterase activity in excised and reconstructed human skin – Biotransformation of prednicarbate and the model dye fluorescein diacetate. European Journal of Pharmaceutics and Biopharmaceutics 84: 374–385.

Küchler, S., Wolf, N. B., Heilmann, S., Weindl, G., Helfmann, J., Yahya, M. M., Stein, C. & Schäfer-Korting, M. (2010): 3D-wound healing model: influence of morphine and solid lipid nanoparticles. J Biotechnol 148, 24-30.

Semlin, L., Monika Schäfer-Korting, M., Claudia Borelli, C. & Korting, H. C. (2011): In vitro models for human skin disease. Drug Discovery Today 16, 3/4: 132-139.

Casati, S. (2013): Non-animal Tests and their Validation Status The EURL-ECVAM Strategy for Skin Sensitisation. EPAA-LRI Skin Sensitisation Training Workshop (http://ec.europa.eu/enterprise/epaa/3_events/3_3_workshops/07-skin-sens-workshop-casati-feb2013_en.pdf)



Glossary:

¹ Keratinocytes: a special skin cell type that makes up 90% of the cells in the epidermis. Keratinocytes produce keratin (horn substance).

² Fibroblasts: Connective tissue cells that primarily produce collagen.

³ T cells: a special immune cell type produced in the thymus (hence the name “T”).

⁴ Surface receptors: molecules on the surface of cells that have a specific shape onto which corresponding foreign substances can dock according to the key-lock principle.

⁵ Local lymph node assay (LLNA): OECD Test Guideline 429. The test substance is applied to the ears of mice. Three days later, the auricular lymph nodes are removed and the cell division id determine. There are two approved variations of this test. In addition to the LLNA, there are tests on guinea pigs (the so-called Buehler test and the guinea pig maximisation test).

⁶ ECVAM, more precisely EURL ECVAM (European Union Reference Laboratory for Alternatives to Animal Testing): European body for the validation of alternatives to animal testing at the European Union Joint Research Centre. (http://ihcp.jrc.ec.europa.eu/our_labs/eurl-ecvam)

⁷ Casati, S. (2013): http://ec.europa.eu/enterprise/epaa/3_events/3_3_workshops/07-skin-sens-workshop-casati-feb2013_en.pdf

⁸ ITS (Integrated Testing Strategy): Approach that integrates different types of data and information into decision-making processes. ITS includes not only information from single tests, test batteries or graded test schemes, but also so-called “weight of evidence” data, data on exposure and population, and a final risk evaluation for a test substance (http://alttox.org/ttrc/emerging-technologies/its/).

⁹ Epidermis: The skin is made up of three layers: epidermis (upper skin), dermis (middle layer) and subcutis (the lowest layer, comprising fatty tissue). The immune cells migrate to the lymph nodes from the epidermis if they have had contact with pathogens.