Abstract
Methodologies in toxicological research presently rely heavily on in vivo approaches. Since ethical considerations forbid human testing in most cases, animal studies are employed as a close depiction of human pathophysiology. Controversy on the value of data obtained from in vivo study is ongoing, especially with regard to translatability. Still, a considerable number of animal-based studies are conducted in basic research, industry, and the pharmaceutical sector, in part required by law. The current situation makes ex vivo and in vitro approaches take a back seat, despite the fact that many such methods are available, and that they can be superior to in vivo experimentation in terms of methodology and quality of results. Implementation of such alternatives can broaden the investigative spectrum and increase chances to receive funding. In addition, the ethical problems of in vivo methods can be averted, especially with regard to the ever-increasing numbers of animal use in research. 1 Many ex vivo and in vitro techniques satisfy the “3R” approach—therefore imply a much needed reduction, refinement, and replacement of animal experimentation. 2 Beginning with this issue, Applied In Vitro Toxicology will feature commentaries on selected alternatives to in vivo study, to highlight scientific and ethical merits. To start, this commentary will focus on the ex vivo technique Precision Cut Lung Slices (PCLS).
Organ slice techniques serve as a link between in vivo and in vitro methodologies and can support the shift away from reliance on animal testing. Experimental refinement is fulfilled because whole animal exposures are not necessary. Organs can be sampled, sliced, and subjected to treatment, which reduces animal discomfort to handling and euthanasia. Apart from the need for an appropriate slicing device, low maintenance standard equipment is needed for preparation and incubation of slices, and the implementation of operating protocols is fairly easy. Even lungs from small animals such as mice yield a high number of slices. These are indeed true technical replicates, thus allowing one to compare toxic responses relative to control or dose alternative in the self-same organ. The presence of multiple technical replicates from a single animal considerably increases the power of the experimentation. Thereby, a definitive reduction of necessary animal numbers can be achieved.
All organ slices, including PCLS, are three-dimensional excerpts of the actual organ, comprising all resident cell types alongside their physiological structural and functional interactions. Allowing one to reserve the microenvironment of the cell, while considering its response to exposure. The possible applications for toxicology research alone are manifold. PCLS have been employed for studies on airway responsiveness, immunotoxicity, neurotoxicity, genotoxicity, and many more.3–6 Commonly, toxicological endpoints comprise airway contractility, cellular viability after exposure to toxicants, analyses of the transcriptome, and histology. Standard cell culture analyses such as Patch Clamp or comet assays have also been shown to be applicable for PCLS. Therefore, PCLS allow the measurement of both, physiological and cell biological endpoints in the same tissue, representing a refinement of experimental design.
The makeup of PCLS permits the investigation of organ-specific responses. Especially for pulmonary toxicology, distinct studies on dose delivery can be conducted, which cannot be accomplished in whole animal experimentation. In many in vivo models of pulmonary exposure there are difficulties in reproducibility and applicability that result from the method of administration. As there is a great deal of comparability of results between PCLS and in vivo approaches, one can see that these methods are suitable to serve as a valid alternative. An exceptional key advantage of the technique is the possibility to use human organ material, thereby discarding questions of translatability and fulfilling the replacement criterion of the 3R.
Naturally, no scientific approach is without limitations. The lack of perfusion and ventilation in PCLS seems to reduce the methods validity for studies of inflammation and effects of airborne noxa. The latter can be addressed by expanding the PCLS technique with an air–liquid-interface setup. Since cellular functions are preserved in PCLS, onset of inflammatory responses can be analyzed, thereby contributing to immunological investigations. Notably, studies of chronic pulmonary diseases are still limited to in vivo approaches, mostly because PCLS viability currently does not exceed 15 days in culture. Efforts are, however, ongoing to prolong slice longevity. Also, suitability of PCLS for investigation of fibrotic tissue responses has already been demonstrated. 7
The implementation of ex vivo organ slices in pharmacological and toxicological research needs to be encouraged, to enhance the scientific value and quality of results. Advanced methods such as PCLS can be a valuable addition to existing protocols and can refine, reduce, and, if applicable, replace animal experimentation.