In this study, we describe the isolation and immortalisation of primary murine alveolar epithelial cells (mAEpC), as well as their epithelial differentiation and barrier properties when grown on Transwell® inserts. Like human alveolar epithelial cells (hAEpC), mAEpC transdifferentiate in vitro from an alveolar type II (ATII) phenotype to an ATI-like phenotype and exhibit features of the air–blood barrier, such as the establishment of a thin monolayer with functional tight junctions (TJs). This is demonstrated by the expression of TJ proteins (ZO-1 and occludin) and the development of high transepithelial electrical resistance (TEER), peaking at 1800Ω•cm2. Transport across the air–blood barrier, for general toxicity assessments or preclinical drug development, is typically studied in mice. The aim of this work was the generation of novel immortalised murine lung cell lines, to help meet Three Rs requirements in experimental testing and research. To achieve this goal, we lentivirally transduced mAEpC of two different mouse strains with a library of 33 proliferation-promoting genes. With this immortalisation approach, we obtained two murine alveolar epithelial lentivirus-immortalised (mAELVi) cell lines. Both showed similar TJ protein localisation, but exhibited less prominent barrier properties (TEERmax ~250Ω•cm2) when compared to their primary counterparts. While mAEpC demonstrated their suitability for use in the assessment of paracellular transport rates, mAELVi cells could potentially replace mice for the prediction of acute inhalation toxicity during early ADMET studies.
A Review of the Contributions of Cross-discipline Collaborative European IMI/EFPIA Research Projects to the Development of Replacement, Reductionand Refinement Strategies
The objective of this review is to report on whether, and if so, how, scientific research projects organised and managed within collaborative consortia across academia and industry are contributing to the Three Rs (i.e. reduction, replacement and refinement of the use of animals in research). A number of major technological developments have recently opened up possibilities for more direct, human-based approaches leading to a reassessment of the role and use of experimental animals in pharmacological research and biomedicine. This report reviews how projects funded by one of the research funding streams, the Innovative Medicines Initiative (IMI), are contributing to a better understanding of the challenges faced in using animal models. It also looks how the results from these various projects are impacting on the continued use of laboratory animals in research and development. From the progress identified, it is apparent that the approach of private–public partnership has demonstrated the value of multicentre studies, and how the spirit of collaboration and sharing of information can help address human health challenges. In so doing, this approach can reduce the dependence on animal use in areas where it has normally been viewed as necessary. The use of a collaborative platform enables the Three Rs to be addressed on multiple different levels, such that the selection of models to be tested, the protocols to be followed, and the interpretation of results generated, can all be optimised. This will, in turn, lead to an overall reduction in the use of laboratory animals.
Traumatic Brain Injury (TBI) remains a significant cause of mortality and morbidity, affecting individuals of all age groups. Much remains to be learned about its complex pathophysiology, with a view to designing effective neuroprotective strategies to protect sublethally injured brain tissue that would otherwise die in secondary injury processes. Experimental in vivo models offer the potential to study TBI in the laboratory, however, treatments that were neuroprotective in animals have, thus far, largely failed to translate in human clinical studies. In vitro models of neurotrauma can be used to study specific pathophysiological cascades — individually and without confounding factors — and to test potential neuroprotective strategies. These in vitro models include transection, compression, barotrauma, acceleration, hydrodynamic, chemical injury and cell-stretch methodologies. Various cell culture systems can also be utilised, including brain-on-a-chip, immortalised cell lines, primary cultures, acute preparations and organotypic cultures. Potential positive outcomes of the increased use of in vitro platforms to study TBI would be the refinement of in vivo experiments, as well as enhanced translation of the results into clinically meaningful neuroprotective strategies for the future. In addition, the replacement of in vivo experiments by suitable in vitro studies would lead to a welcome reduction in the numbers of animal procedures in this ethically-challenging field.
Jodie Melbourne, Patricia Bishop, Jeffrey Brown and Gilly Stoddart
In 2015, the PETA International Science Consortium Ltd. was awarded the Lush Training Prize for its broad approach to education and training on the effective use of human-relevant, non-animal research techniques. The prize was awarded for work that included hosting workshops and webinars, initiating in-person training sessions and developing educational resources. The Consortium works closely with industry and regulatory agencies to identify and overcome barriers to the validation and use of alternatives to animal testing, by using an approach that identifies, promotes and verifies the implementation of these methods. The Consortium's recent activities toward replacing tests on animals for nanomaterials, pesticides and medical devices, are described, as examples of projects with broad applicability aimed at large-scale regulatory change.
Rabea Graepel, David Asturiol, Pilar Prieto and Andrew P. Worth
A survey was carried out to explore opportunities for waiving mammalian acute systemic toxicity tests. We were interested in finding out whether data from a sub-acute toxicity test could be used to predict the outcome of an acute systemic toxicity test. The survey was directed at experts in the field of toxicity testing, and was carried out in the context of the upcoming 2018 final registration deadline for chemicals under the EU REACH Regulation. In addition to the survey, a retrospective data analysis of chemicals that had already been registered with the European Chemicals Agency, and for which both acute and sub-acute toxicity data were available, was carried out. This data analysis was focused on chemicals that were administered via the oral route. The answers to the questionnaire showed a willingness to adopt waiving opportunities. In addition, the responses showed that data from a sub-acute toxicity test or dose-range finding study might be useful for predicting chemicals that do not require classification for acute oral toxicity (LD50 > 2000mg/kg body weight). However, with the exception of substances that fall into the non-classified category, it is difficult to predict current acute oral toxicity categories.
Elisabeth H. Ormandy
This interview-based study examined the diversity of views relating to the creation and use of genetically-engineered (GE) animals in biomedical science. Twenty Canadian participants (eight researchers, five research technicians and seven members of the public) took part in the interviews, in which four main themes were discussed: a) how participants felt about the genetic engineering of animals as a practice; b) governance of the creation and use of GE animals in research, and whether current guidelines are sufficient; c) the Three Rs (Replacement, Reduction, Refinement) and how they are applied during the creation and use of GE animals in research; and d) whether public opinion should play a greater role in the creation and use of GE animals. Most of the participants felt that the creation and use of GE animals for biomedical research purposes (as opposed to food purposes) is acceptable, provided that tangible human health benefits are gained. However, obstacles to Three Rs implementation were identified, and the participants agreed that more effort should be placed on engaging the public on the use of GE animals in research.
The Use of a Decision Tree Based on the Rabies Diagnosis Scenario, to Assist the Implementation of Alternatives to Laboratory Animals
Vanessa Carli Bones and Carla Forte Maiolino Molento
Brazilian federal legislation makes the use of alternatives mandatory, when there are validated methods to replace the use of laboratory animals. The objective of this paper is to introduce a novel decision tree (DT)-based approach, which can be used to assist the replacement of laboratory animal procedures in Brazil. This project is based on a previous analysis of the rabies diagnosis scenario, in which we identified certain barriers that hinder replacement, such as: a) the perceived higher costs of alternative methods; b) the availability of staff qualified in these methods; c) resistance to change by laboratory staff; d) regulatory obstacles, including incompatibilities between the Federal Environmental Crimes Act and specific norms and working practices relating to the use of laboratory animals; and e) the lack of government incentives. The DT represents a highly promising means to overcome these reported barriers to the replacement of laboratory animal use in Brazil. It provides guidance to address the main obstacles, and, followed step-by-step, would lead to the implementation of validated alternative methods (VAMs), or their development when such alternatives do not exist. The DT appears suitable for application to laboratory animal use scenarios where alternative methods already exist, such as in the case of rabies diagnosis, and could contribute to increase compliance with the Three Rs principles in science and with the current legal requirements in Brazil.
A Training Course on Laboratory Animal Science: An Initiative to Implement the Three Rs of Animal Research in India
Kunal Pratap and Vijay Pal Singh
There is a current need for a change in the attitudes of researchers toward the care and use of experimental animals in India. This could be achieved through improvements in the provision of training, to further the integration of the Three Rs concept into scientific research and into the regulations of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). A survey was performed after participants undertook the Federation of European Laboratory Animal Science Associations (FELASA) Category C-based course on Laboratory Animal Science (in 2013 and 2015). It revealed that the participants subsequently employed, in their future research, the practical and theoretical Three Rs approaches that they had learned. This is of great importance in terms of animal welfare, and also serves to benefit their research outcomes extensively. All the lectures, hands-on practical sessions and supplementary elements of the courses, which also involved the handling of small animals and procedures with live animals, were well appreciated by the participants. Insight into developments in practical handling and welfare procedures, norms, directives, and ethical use of laboratory animals in research, was also provided, through the comparison of results from the 2013 and 2015 post-course surveys.
Róber Bachinski, Thales Tréz, Gutemberg G. Alves, Rita de C.M. Garcia, Simone T. Oliveira, Luciano da S. Alonso, Júlio X. Heck, Claudia M.C. Dias, João M. Costa Neto, Alexandro A. Rocha, Valeska R.R. Ruiz and Rita L. Paixão
Humane education and the debate on alternatives to harmful animal use for training is a relatively recent issue in Brazil. While animal use in secondary education has been illegal since the late 1970s, animal use in higher science education is widespread. However, alternatives to animal experiments in research and testing have recently received attention from the Government, especially after the first legislation on animal experiments was passed, in 2008. This article proposes that higher science education should be based on a critical and humane approach. It outlines the recent establishment of the Brazilian Network for Humane Education (RedEH), as a result of the project, Mapping Animal Use for Undergraduate Education in Brazil, which was recognised by the 2014 Lush Prize. The network aims to create a platform to promote change in science education in Brazil, starting by quantitatively and qualitatively understanding animal use, developing new approaches adapted to the current needs in Brazil and Latin America, and communicating these initiatives nationally. This paper explores the trajectory of alternatives and replacement methods to harmful animal use in training and education, as well as the status of humane education in Brazil, from the point of view of educators and researchers engaged with the network.
To Be Credible, Success in “Reducing the Use of Animals in Scientific Research” Must Involve the Use of Fewer Animals
Much worthy effort on the Three Rs is under way in the UK, but its promise of a reduction in laboratory animal use will only be credible and laudable when the number of procedures and animals are progressively and permanently reduced.