3Rs-Centre Utrecht Life Sciences

Faculteit Dierengeneeskunde
November 2016
In vitro test strategies reduce, replace and refine animal experiments in neurotoxicity screening

Remco Westerink is university teacher at the Institute for Risk Assessment Sciences (IRAS) and head of the Neurotoxicology Research Group. He investigates the effect of substances on the nervous system, with special attention to new psychoactive substances (NPS). He is one of the first scientists worldwide to use a multi-well microelectrode array (mw-MEA) to screen (psychoactive) substances for neurotoxicity. Thanks to this in vitro method, less laboratory animals are needed to gain the same amount of data.

Within the field of substance testing, many organizations still depend on in vivo experiments to screen for neurotoxic effects1. However, from an ethical point of view, but also from a financial and time-saving perspective, there is a clear need for in vitro test strategies. This made Remco Westerink and his group search for alternatives in neurotoxicity testing, which has led him to the recently developed multi-well microelectrode arrays (mwMEA)2. Using this method, a few cortical cells can develop into spontaneous active functional networks. This way, the neurotoxic effect of substances can be studied in an in vitro environment that includes all receptors, ion channels and other components that are normally present in the brain in an in vivo experiment.

How does it work?
The MEA has been developed to study neuronal activity in vitro by measuring local field potentials of electrically active neuronal cells. In order to test the effects of a particular substance on neuronal activity, rat primary cortical cells are seeded on a 48-well plate. On this plate, the cells start interacting, as if it were an in vivo neural network. At the optimum of the developmental phase, one well contains about 100.000 neurons. The scientists are confident the cells form an integrated network, since they fire in synchrony. Once the network is in this optimal condition, between 10 to 14 days after seeding, the substance of interest is applied in different concentrations to the cells. The field potentials in each well are read out and plotted against baseline activity, resulting in the desired concentration-response graphs.  

Demonstration of a multi-well micro-electrode array and its recordings of neuronal activity.

Towards a future without the use of animals
Although animals are still needed as tissue donor for this method, fewer are needed than before. As an illustration, when single cell patch clamps were used to test for neurotoxicity, only 5 cells could be tested in one day. With mw-MEA, using the same number of animals, substantially more data points can be generated; the number of animals originally used to measure 5 cells with the patch-clamp technique, can now be used to induce the development of neural networks on ten 48-wells plates. Furthermore, instead of exposing an animal to a possible toxicity hazard, like in the mouse bioassay3, only its neural network is exposed. Therefore, the use of this technique also attributes to refinement, and has the potential to replace bioassays in general. In addition, the research group tries to share the remaining tissues of the animals as much as possible, since they only need the cortex. Collaborations with other groups on the Utrecht Life Sciences campus have been established, resulting in a more efficient use of the laboratory animals, thus realizing a reduction in the total number of animals.

Another asset of this in vitro method is that it has a wide range of possible applications. It can be used to test for food safety, but may also be used to screen real environmental samples, like, for example, water for neurotoxic pollutants. It can also be very useful in specific research fields, like, for example, in pain research, where the experiments by definition cause discomfort in animals. It would be a great progress in 3Rs-application, if these experiments could be carried out in an in vitro setting.

Unfortunately, a cortical primary culture from the rat is still needed for this method. This means that the translational value of the screening outcomes with this tissue is still a topic of discussion. Therefore, the group is looking for large data sets of human concentration-response data for certain compounds, in order to validate the rat-derived data. Additionally, the group is going into another exiting direction: to increase the relevance of this method, Westerink’s group also started to use human induced pluripotent stem cell (iPSC)-derived neurons4.

IRAS is an interfaculty research institute within the faculties of Veterinary Medicine, Medicine and Sciences of Utrecht University. IRAS provides education and research on the human health risks of exposure to potentially harmful agents in the environment, at the workplace and through the food chain. Effects on ecosystems are also considered.

  1. European Commission, E., Commission Regulation (EU) No. 15/2011. Offic. J. Eur. Union 2011, L6/3–L6/6.
  2. Hondebrink, L, Verboven, A.H.A., Drega, W S, Schmeink, S, de Groot, Martje, van Kleef, R G D M, Wijnolts, F M J, de Groot, Aart, Meulenbelt, J & Westerink, R H S (2016). Neurotoxicity screening of (illicit) drugs using novel methods for analysis of microelectrode array (MEA) recordingsNeurotoxicology, 55, (pp. 1-9)
  3. Stewart, I, McLeod, C (2014). The laboratory mouse in routine food safety testing for marine algal biotoxins and harmful algal bloom toxin research: past, present and future. J AOAC Int. 97(2): (pp.356-72)
  4. Tukker, Anke M, de Groot, Martje W G D M, Wijnolts, Fiona M J, Kasteel, Emma E J, Hondebrink, Laura & Westerink, Remco H S (2016). Is the time right for in vitro neurotoxicity testing using human iPSC-derived neurons? ALTEX-Alternatives to Animal Experimentation