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Alternative CAM: chicken eggs to precede mouse experiments

Researchers who test whether in vitro results can be transferred to living organisms prefer to use mice, rats or rabbits. However, the step from cell culture to animal experiments is often taken far too soon, said Karin Kunzi-Rapp, biologist and dermatologist from Ulm. Many questions can be answered using the chorioallantoic membrane (CAM) of fertilised chicken eggs, an alternative model to animal experiments that provides rapid results and only costs 50 cents per egg. Moreover, it does not raise any ethical concerns.

A tumour has developed on the chorioallantoic membrane of an incubated chicken egg. © ILM

The chorioallantoic membrane (CAM) is a vascular membrane found in chicken eggs. It is very thin, consists of three different layers and is criss-crossed with numerous blood vessels. This membrane corresponds to the placenta in mammals, said Kunzi-Rapp, who did her medical degree thesis at the ILM, an institute associated with Ulm University. Her degree thesis focused on assessing the suitability of the CAM model for photodynamic therapy. She learned to handle the model in Haifa, Israel, where she did her degree thesis as part of a project of the German-Israeli Society.

The experiments very quickly showed that the CAM is well suited as a model system for photodynamic therapy. It has been shown that tumour cells that are seeded on the CAM developed into three-dimensional tumours, thereby confirming previous in-vitro results. This finding, however, was not enough to satisfy Karin Kunzi-Rapp's curiosity. She went on to focus on finding further possibilities for application of the model that led to the discovery of a broad range of applications and the establishment of the chorioallantoic membrane as an alternative model to replace animal experiments.

Although the CAM model has been known for a long time, some people remain unaware of its advantages

The CAM model has been known since the end of the 19th century. It was originally designed for use in embryology, was then discovered as a model for transplantations and finally fell into oblivion. It is still used for the production of vaccines and in the field of virology for the cultivation of viruses. Kunzi-Rapp also tells us that the model was rediscovered in the 1970s by an American scientist as a tumour and vascular model. When she assessed the CAM model for its suitability as an alternative to animal experiments and wanted to publish her results, nobody was really interested in her findings. “The majority of people believed that the CAM was not close enough to the way the animal model worked.” However, in the meantime, public authorities have started to promote the use of CAM as an alternative to animal experiments. “Nevertheless, the CAM model is not yet known to everyone,” said Karin Kunzi-Rapp.

Between in-vitro and animals

Dr. Karin Kunzi-Rapp has already established the CAM model among researchers at the ILM and the University of Ulm. © ILM

The chorioallantoic membrane is somewhere between two- and three-dimensional spheroid models used in in vitro studies and animal models. The model does not involve the embryo itself, but only the outer, strongly vascularised membrane. The growth of tumours can be triggered on the surface of the CAM and tissue can be transplanted. Therefore, the CAM enables three-dimensional work to be carried out on a functional vascular system. "In principle, this is the same as in animal experiments," but is not regarded as such in the first phase of incubation (day 1 to 10). Therefore, it is not necessary to apply for permission to carry out animal experiments. According to Animal Protection Law in Germany, animal embryos are not regarded as animals and experiments involving the CAM do not have to be registered. The German Animal Protection Law regards chickens as animals only when they hatch after 21 days.

The chorioallantoic membrane does not contain any nerves, so the embryo does not experience any pain. The CAM is also ideally suited as an animal replacement model because, in common with immunoincompetent mice, it does not have an immune defence system. This is a huge advantage for researchers who are planning to transplant and grow any tissue on the membrane. "This is the great advantage: I can work with human cells and tissue."

For example, the method works well for human skin. Karin Kunzi-Rapp and her colleague Angelika Rück from the ILM received the Felix Wankel Animal Protection Research Prize in 1997 for their findings that newly removed human skin can be transplanted to the CAM. Kunzi-Rapp showed that the vessels of human skin connect up with the vascular system of the chicken embryo and that the vessels of the human skin remain human, while the blood flowing through them originates from the chicken embryo.

A model that is easy to work with

Cell suspensions can be applied easily to the membrane. © ILM

In contrast to experimental animals, researchers using incubated chicken egg have far less work. The only thing they have to do is to place the egg into a 38-degree incubator. After four days, they drill a small hole into the hard eggshell. If the egg has been fertilised, the beating heart of the transparent embryo and the membrane can be clearly discerned. Unlike the production of vaccines, the CAM model does not need to be sterile, because the egg is able to fend off bacteria. Fungal infections occur, but are relatively rare. The chicken embryos are "killed" after the experiments by cutting the vessels running from the chorioallantoic membrane to the embryo. This leads to an immediate "volume deficiency shock" and the embryos die quickly.

The CAM model only allows short-term investigations to be carried out. Researchers use the model between the sixth and 11th day of incubation. The chicken embryo starts to develop an immune system from day 12. The CAM is used for the investigation of angiogenesis, especially in tumour research, which is in principle completed on day 10 after fertilisation.

CAM has replaced controversial rabbit experiments

In the meantime, the cosmetics industry, seeking to test its products on human skin, has been showing a growing interest in the CAM model due to the fact that the European Cosmetics Directive has banned animal experiments. Therefore, the CAM has replaced the highly controversial Draize eye irritancy test used to assess the irritation potential of cosmetics by placing liquid, flake and powdered substances into the eyes of rabbits.

Karin Kunzi-Rapp continues to promote the CAM model, for example at the University of Stuttgart-Hohenheim where she lectures once a semester on alternatives to animal experiments. She calls on researchers to refrain from immediately thinking of carrying out animal experiments once initial cell culture experiments have been carried out successfully. She asks them to bear in mind the possibility of using the chicken egg model for clarifying certain questions, and to only move on to animal experiments once a certain substance has proved that it meets future requirements. She advocates a three-dimensional vascular tissue model for testing all necessary parameters before switching to animal models.

Ulm researchers are now focusing on the intermediary solution provided by chicken eggs

At the University and University Hospital of Ulm, many research-based departments are now beginning to use chicken eggs rather than rats, mice or rabbits. A kind of “core facility” has been established at the University of Ulm, which works with all the research-based departments, including the departments of paediatrics, urology, naturopathy, internal medicine and pharmacology.

According to Karin Kunzi-Rapp, the CAM model is perfectly suited for a broad range of applications. She also points out that many results obtained with chicken eggs have subsequently been confirmed in other animals, for example the pharmacokinetics in chicken eggs is the same as in animals and humans, as investigations carried out in cooperation with the Department of Naturopathy at the University of Ulm have shown. “In principle, we can investigate as many things with the CAM as we can with mice, with the only difference that the CAM model requires much less time,” said Kunzi-Rapp summarising her experiences.

Many researchers who have applied for the authorisation to carry out animal experiments and been refused often contact Karin Kunzi-Rapp for help. As the authorities often require intermediary steps to be carried out before moving on to animal experiments, entire research groups from all over Germany have come to Ulm to learn about and use the chorioallantoic membrane model. Ten years ago, there were no more than two industrial research groups that used the CAM model.

Kunzi-Rapp attaches great importance to independence

During her biology studies at the University of Stuttgart-Hohenheim, Karin Kunzi-Rapp realised that she wanted to work in human medicine research. Therefore, she abandoned her PhD thesis in the field of biochemistry at the University of Ulm and took up medicine. The dermatologist now works at the Department of Dermatology at Ulm University Hospital and heads up the Laser Therapy Centre at the Ulm-based ILM, where she treats patients transferred to her from the Department of Dermatology. At present, her major focus is on clinical studies because a growing number of laser manufacturers are interested in using the lasers for cosmetic applications, which eventually end up in Kunzi-Rapp’s department at the ILM.

Kunzi-Rapp has repeatedly rejected lucrative offers from industry or beauty clinics. “I am more a researcher than anything else,” said Kunzi-Rapp explaining that she is more interested in finding out why a certain laser application works as it does. She also attaches greater importance to independence than to money, which is also why she uses some of her “spare” time to work in the neurofibromatosis consultation hour at the Ulm University Hospital, treating patients from all over Germany.

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