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

虽然传统上基本的生命过程是在整个生物体水平上(即‘体内’)研究，但近年来，由于经济、伦理和法律原因，人们非常重视使用体外培养的细胞、组织或器官，即所谓的‘体外’系统。在这方面，近几十年来，由于人口增长、工业化和消费者需求的增加，生活系统面临的环境压力或污染物有所增加。消费者或工业活动产生的这些污染物中的许多最终进入了水环境。暴露在这种压力下会导致许多疾病的增加，并可能影响野生物种。为了评估这种污染物的潜在有害影响，国际法要求化学品或消费品在动物身上进行测试。虽然在减少生物医学研究中使用的动物数量方面取得了进展，但仍需要使用鱼类来评估污染物的潜在毒性影响。已提议使用细胞或组织模型进行基于鱼类的研究，其好处是只需要几只捐献动物。然而，目前还不清楚这些模型能否很好地预测使用整个动物时的反应。因此，人们不愿接受这些作为一种真正的替代方案。传统的悬浮或单层体外培养模型也存在缺陷(例如，它们在实验室中存活时间较短，一些功能在动物外不起作用)，使它们不适合长期研究。基于广泛使用的哺乳动物模型，包括我们自己的团队，我们已经从鱼的肝细胞中开发出一种专门的培养物，它可以‘模拟’整条鱼组织的反应。这些球状结构被称为“球体”，其寿命是现有单层(单层)细胞的十倍。我们已经证明，它们能够以一种与整条鱼直接相当的方式吸收和代谢化学物质，这表明它们可以作为整个动物使用的真正替代品。因为这些椭圆形细胞只由少数动物制备，而不是通常需要数百只动物来研究生物医学程序和评估毒性。它们的体积小意味着需要更少的材料和更小的设施，使它们进行研究的成本更低。由于从一条鱼身上可以收获来自几个不同器官的数十亿个细胞，这意味着更少的鱼将被用于研究，而那些不直接用于实验的鱼将只在牺牲后收获它们的组织。我们的目标是改进这个模型，超越最先进的技术来测试包含不同细胞或组织类型的鱼的共培养组合(即不同的细胞类型一起生长)的假设。(鳃、内脏和肝脏)为活鱼研究提供了一种有效的替代模型，从而创造了一种“虚拟鱼类”替代方案。这需要在细胞或分子水平上建立关键的生物反应，称为“生物标记物”，它们已经在一系列人类疾病的诊断、预后和治疗中得到了很好的证实。利用这些生物标志物，我们的目标是阐明鱼类对这种应激反应的机制和生理功能，包括长期暴露于与环境相关的化学物质。然后，我们的目标是进行强有力的比较，以评估该模型是否足够敏感，可以被视为全鱼研究的适当替代。该提案还旨在在产业界、学术界和国内外的利益相关者之间建立强有力的联系，以共享知识库，并可能获得更广泛的监管批准，作为动物试验的真正替代方案，以继续我们支持3R(减少、改进和替换)倡议的承诺。

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.