Moving up a dimension: 3D in vitro models as effective alternatives to live fish studies

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Whilst traditionally fundamental life processes are studied at whole organism level (i.e. 'in vivo'), in recent years for economic, ethical and legal reasons, there has been much emphasis on the use of cells, tissues or organs which are grown outside the body in plastic dishes or flasks, the so called 'in vitro' system. In this context, in recent decades due to population growth, industrialisation and increasing consumer demands, there has been an increase in the environmental stress or contaminants on the living systems. Many of these contaminants resulting from consumers or industrial activities end-up in the aquatic environment. Exposures to such stresses lead to an increase in many diseases and can impact wild species. In order to assess the potential detrimental impact of such contaminants, international law requires chemicals or consumer products to be tested on animals. While advances have been made to reduce the number of animals used in biomedical research, the use of fish is still required for assessing the potential toxic effects of contaminants. The use of cell or tissue-based models has been proposed for fish-based studies, with the benefit of requiring only a few donor animals. It is however not clear how well these models could predict the responses that are seen when using whole animals. There is therefore reluctance to accept these as a real alternative. The conventional suspension or single layer in vitro culture models also suffer from drawbacks, (e.g. they live for a short period in the laboratory, and some functions just do not work outside of the animal) making them unsuitable for long term studies. Based on widely used mammalian models, including by our own team, we have developed a specialised culture from liver cells of fish that 'mimic' the responses of tissues in whole fish. These balls like structures called 'spheroids' live ten times longer than the existing single layer (monolayer) cells. We have demonstrated that they are able to take-up and metabolise chemicals in a way that is directly comparable to whole fish, suggesting that they could be used as a real alternative to whole animal usage. As these spheroid cells are prepared from only a handful of animals, as opposed to hundreds of animals typically needed to study biomedical procedures and to assess toxicity. Their small sizes mean that less material and smaller facilitates are required, making them less expensive to conduct research. Since billions of cells from several different organs can be harvested from a single fish means that far fewer fish will be used in research, and those that are will not be used directly in experiments, only their tissues harvested after sacrifice.We aim to progress this model, beyond-the state-of art technologies to test the hypothesis that a combination of co-cultures (i.e. different cell types grown together) containing different cell or tissue types of fish (viz. gill, gut and liver) represents an effective alternative model to live fish studies, thus creating a 'virtual fish' alternative. This requires establishment of key biological responses at cellular or molecular levels called 'biomarkers', which are already well established in diagnosis, prognosis and treatments of a range of human diseases. Using these biomarkers, we aim to elucidate the mechanisms and physiological functions by which fish respond to such stresses including following chronic exposures to environmentally relevant chemicals. We then aim to make robust comparisons to assess whether the model is sensitive enough to be considered as an appropriate replacement to whole fish studies. The proposal also aims to build strong links between industry, academia and stake holders, both nationally and internationally to share the knowledge base as well as to potentially gain wider regulatory approval as 'real' alternatives to animal tests to continue our commitment to supporting the 3R's (Reduction, Refinement & Replacement) initiative.