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

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.

The proposal aims to probe the primary hypothesis that a co-culture of 3D in vitro cultures (viz. liver-spheroids and gill / gut epithelia) from rainbow trout (Oncorhynchus mykiss) can represent an effective alternative model to live fish studies. Upon establishing standard conditions for the growth and maintenance of co-cultures, we aim to:

1.Fully characterise the system and further define the culture conditions to ensure basic functionality is maintained and viability is prolonged.

2.Establish baseline values and compare key proteomic and recognised molecular biomarkers in in vitro preparations and compare the values with available in vivo data.

3.Conduct a limited set of in vivo studies to provide material directly relevant to and from the same genetic stock as the cells used in vitro in order to confirm the comparability from in vivo to in vitro.

4.Determine the functionality of spheroids alone or as co-cultures with respect to basic bio-transformation pathways and metabolite formation following exposures to different chemical agents, (e.g. reference genotoxic chemicals: polycyclic aromatic hydrocarbons; environmentally relevant pharmaceuticals: non-steroidal anti-inflammatory, beta-blocker and selective serotonin re-uptake inhibitors; and environmentally relevant metals: copper).

5.Compare the comparative physiology (oxygen uptake, bio-energetics, metabolism and excretion rates) of the systems in response to 'normal' control conditions and exposure to environmentally relevant chemicals.

6.Evaluate 'mixture' toxicity from a 'toxicogenomic' perspective in both isolated tissue preparations and co-cultures.

7.Measure the bioconcentration of selected chemicals in all tissue types following chronic exposure to test chemicals using cutting edge analytical techniques.

8.Communicate the methodologies and findings to key industrial, academic and stakeholder groups.

The outcome of this interdisciplinary proposal will make a significant difference to diverse groups of people in the wider scientific community in a variety of ways:

In terms of the applied science, one particular example that has great relevance to industry is the advent of the European chemical registration and approval (REACH ) that has lead to the pre-registration of >140,000 compounds, with >30,000 of these requiring significant in vivo testing. A large proportion of these will require the assessment of the bioaccumulation potential of these compounds via standard regulatory OECD tests such as the fish bioaccumulation test 305. In 2011 (according to the UK Government) 58,908 fish were reported as used in toxicology studies in the UK alone. Testing in other European countries, have similar rates. The majority of these fish are likely to be rainbow trout.

The requirement of extensive facilities, large amounts of test compound, often radio-labelled and significant numbers of fish clearly demonstrates the ethical and financial reasons that are preventing the rapid assessment of these compounds, which pose an unknown risk to the environment until they can be effectively tested. This information is essential for Environmental Risk Assessment and therefore the need for reliable, alternative testing methods are essential if these thousands of compounds are to be assessed without requiring millions of fish. As this field grows, and we help other groups to work with our model, we believe the studies will be fundamental and seminal work that will receive much attention and through open access publication and freely available protocols that will deliver well cited influential work.

We envisage that the developed model will serve as a spring board to enable many groups to conduct effective, small scale, low cost, high content, and higher throughput alternatives to study biological processes at whole organism level under in vitro conditions. Our model will offer a new, novel research tool for answering key scientific questions regarding an animal's physiology, that may have been previously too difficult to carry out with whole fish. The fact that these micro-tissues reform the organs outside of the fish means that researchers can see how cells communicate and interact using microscopic and molecular tools. This model could serve as spring board to stimulate design and developments of new investigations in the uptake and elimination of nutrients; cellular transport; immunity; disease progression; organ culture; signalling; cell biology such as cycling, division, replication, apoptosis and protein expression.

This project will provide unique understanding of the fundamental mechanisms, biochemical characteristics and extent of functionality of fish gill, gut and liver tissues both in life and in culture. These data will be of value to a range of fish biologists, and we anticipate that detailed information and understanding of this level of detail will quickly enter the teaching curricular of both fish and general biology.

The fully developed and validated model will be promoted for potential regulatory acceptance as true alternative to live fish bioaccumulation tests. Once accepted this would be necessarily widely taken up by many commercial laboratories in order to meet European and indeed worldwide legislation.

The research team is extensive expertise in disseminating research findings to School children, general public through outreach activities and popular articles in local media and in BBSRC and NERC journals. The PI has also experience in being invited by local BBC (radio and TV) and BBC radio five live as expert commentator. This would enhance not only the public understanding of the BBSRC funded science but will also encourage younger generation for a scientific career.