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

提升一个维度：3D 体外模型作为活鱼研究的有效替代方案

• 批准号: BB/L01016X/1
• 负责人: Awadhesh Jha
• 金额: $ 56.52万
• 依托单位: Plymouth University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FL01016X%2F1
• 关键词: Moving; up; dimension; 3D; vitro

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.

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”）进行研究的，但近年来由于经济、伦理和法律的原因，已经非常重视使用在塑料培养皿或烧瓶中在体外生长的细胞、组织或器官，即所谓的“体外”系统。在这种情况下，近几十年来，由于人口增长、工业化和消费者需求的增加，对生命系统的环境压力或污染物有所增加。许多来自消费者或工业活动的污染物最终进入水生环境。暴露于这种压力会导致许多疾病的增加，并可能影响野生物种。为了评估这些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。已经有人提议使用基于细胞或组织的模型进行基于鱼类的研究，其好处是只需要几只供体动物。然而，目前尚不清楚这些模型在多大程度上可以预测使用整个动物时所看到的反应。因此，人们不愿意接受这些作为真实的替代办法。常规的悬浮液或单层体外培养模型也具有缺点（例如，它们在实验室中存活时间短，并且一些功能在动物体外不起作用），使得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，我们已经开发出一种专门的培养物，从鱼的肝细胞中“模仿”整个鱼的组织反应。这些被称为“球状体”的球状结构比现有的单层（单层）细胞寿命长十倍。我们已经证明，它们能够吸收和代谢化学物质的方式是直接可比的整个鱼，这表明他们可以作为一个真实的替代整个动物的使用。由于这些球状体细胞仅从少数动物中制备，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。它们的小尺寸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。由于可以从一条鱼身上收获来自几个不同器官的数十亿个细胞，这意味着用于研究的鱼要少得多，而且那些鱼不会直接用于实验，只会在牺牲后收获它们的组织。超越最先进的技术来测试假设，（即不同的细胞类型生长在一起）含有不同的细胞或组织类型的鱼（即鳃、肠和肝）代表了活鱼研究的有效替代模型，从而创造了“虚拟鱼”的替代方案。这需要在细胞或分子水平上建立关键的生物反应，称为“生物标志物”，这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建立。利用这些生物标志物，我们的目标是阐明鱼类对这种压力的反应机制和生理功能，包括长期暴露于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，被认为是一个适当的替代全鱼的研究。该提案还旨在在国内和国际上建立行业，学术界和利益相关者之间的紧密联系，以共享知识基础，并可能获得更广泛的监管批准，作为动物试验的“真实的”替代品，以继续我们支持3R（减少，改进和替代）倡议的承诺。

项目成果

Fish cell lines in 3-dimensional (3D) cultures in ecotoxicological studies: is there oxygen in the middle?

生态毒理学研究中三维 (3D) 培养的鱼细胞系：中间有氧吗？

• 发表时间: 2015
• 作者: Langan L

Can a cell line behave like native tissue in 3-dimensional form? A case study using RTgutGC as a model for copper uptake and metabolism

细胞系可以表现得像 3 维形式的天然组织吗？

• 发表时间: 2015
• 作者: Langan L

3D in vitro models as effective alternatives to live fish studies poster presentation

3D 体外模型作为活鱼研究海报演示的有效替代品

• 发表时间: 2016
• 作者: Baron MG

Pharmaceutical Metabolism in Fish: Using a 3-D Hepatic In Vitro Model to Assess Clearance.

• DOI: 10.1371/journal.pone.0168837
• 发表时间: 2017
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Baron MG;Mintram KS;Owen SF;Hetheridge MJ;Moody AJ;Purcell WM;Jackson SK;Jha AN
• 通讯作者: Jha AN

Fish in a dish: Replacing animal numbers in aquatic ecotoxicology

盘子里的鱼：取代水生生态毒理学中的动物数量

• 发表时间: 2015
• 作者: Baron M