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Colon of a patient with ulcerative colitis, stained with CD19 (red), CD3 (green), Eomes (dark blue), CD34 (cyan), EpCAM (magenta) and CD103 (yellow), imaged with MELC. Copyright: A. Hauser


Less animal experiments with multiplex microscopy

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Colon of a patient with ulcerative colitis, stained with CD19 (red), CD3 (green), Eomes (dark blue), CD34 (cyan), EpCAM (magenta) and CD103 (yellow), imaged with MELC. Copyright: A. Hauser
Colon of a patient with ulcerative colitis, stained with CD19 (red), CD3 (green), Eomes (dark blue), CD34 (cyan), EpCAM (magenta) and CD103 (yellow), imaged with MELC. Copyright: A. Hauser

In the field of immunodynamics, researchers at the Charité are trying to understand the complex defence reactions of the immune system. Until a few years ago, Prof. Anja Hauser's research group used tissue sections from mice for this purpose. This has now changed - also with the help of support from Charité 3R. The "Multiplex Microscopy" project is a successful example of the fact that "Replace" and "Reduce" are possible in certain areas of research.

If you want to know how the immune system reacts to stimuli, it is not enough to just look at the immune cells. This is because in defence reactions - whether caused by an infectious pathogen or in an autoimmune reaction - a dynamic interplay of different cell types sets in. This is necessary so that an immune reaction can start, continue and also be regulated.

Scientists in the field of immunodynamics are therefore interested in the interactions of the different cell types in tissues. In order to understand the physiological process of a natural defence reaction as well as a misdirected immune response, they have to find out which cell types are actually involved in the process. 

Three-digit savings per experiment 
Only a few years ago, Prof. Anja Hauser's team used conventional histological examination methods - and practically only tissue sections from mice. Until the professor of immunodynamics received funding from Charité 3R in 2019. In the project, Hauser and her team were able to further develop "multiplex microscopy (MELC)" so that considerably more cells can be examined on one tissue section. Ergo, fewer mice are needed for the experiments. In quite a few projects, the number of animals saved is in the three-digit range, as Anja Hauser reports. 

Two thirds of the trials are animal-free today 
But that is by no means all: the researchers were also able to use the funding to buy antibodies and reagents for "alternatives". "The funding opened the door for us to use human tissue instead of animal tissue sections," Anja Hauser tells us. "So we were not only able to reduce the number of animals needed, but even replace a large part." The bottom line to date is that the researchers conduct about two-thirds of all experiments on human tissue. "Replace was a major goal of the project, along with Reduce, and both are still working today."

Multiplex microscopy involves labelling antibodies with fluorescent dye and placing them on the tissue. This allows cells carrying the molecule matching the antibody on their surface to be identified and located. The dye is then bleached out and the next marker can be placed on top. The whole process can be repeated as often as desired - usually 60 to 100 markers per experiment. In contrast, with the conventional examination methods, only four parameters, i.e. markers, could be measured per experiment - which explains the higher number of tissues required.

After the experiments, the individual images are superimposed with the help of a computer, so that a kind of mosaic of the entire tissue is created. Specially developed AI algorithms help to find the cells in the tissue composite again, to track down their neighbours and to show how the cells influence each other. "We have further developed the algorithms so that we can make statistically sound statements about the localisation and interaction of the individual cells," Hauser emphasises. 

Algorithms allow deep insights into cell networks 
Powerful computers were purchased with the Charité 3R funding and used to train image analysis algorithms to recognise individual cells in complex cell networks - something that would probably take months to years to do manually. Machine learning or artificial intelligence has thus decisively improved immunodynamic analyses. Researchers are now able to analyse cell types in depth, which was previously impossible.

The fact that further development of the technology began even before the Corona pandemic was a happy coincidence. Among other things, the research team was able to examine lung tissue from deceased Covid 19 patients with the new method and find out, for example, that Covid-related pulmonary fibrosis is an immune-mediated disease similar to idiopathic pulmonary fibrosis. That is, not because of the virus itself, but because of sustained immune activation, the lung tissue is remodelled into solid, scar tissue. "Thanks to the funding, we had already experimented with human tissue from operations before and knew that our method worked," says Hauser. "This experience then came in handy during the pandemic and we were then able to react very quickly."

Pathologists contribute human tissue 
The team still benefits from the contacts to surgeons and pathologists today. After all, a lot of human tissue is needed for the trials. Hauser, who also heads a working group at the German Rheumatism Research Centre, says she only built up a corresponding network through the project funding. Since then, she has been supplied by colleagues with tissues that she needs for her investigations - for example, chronically inflamed intestinal tissue or tumour tissue. 

(Text: Beatrice Hamberger)


AG Hauser

Charité 3R Förderlinie "Adding 3R Value": Multiplex-Mikroskopie in der Immunologie


Charité 3R

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