in vitro models

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In Vitro Models as a Platform to Investigate Traumatic Brain Injury

Ashwin Kumaria

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.

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In Vitro Exposure Systems and Dosimetry Assessment Tools for Inhaled Tobacco Products: Workshop Proceedings, Conclusions and Paths Forward for In Vitro Model Use

Holger Behrsing, Erin Hill, Hans Raabe, Raymond Tice, Suzanne Fitzpatrick, Robert Devlin, Kent Pinkerton, Günter Oberdörster, Chris Wright, Roman Wieczorek, Michaela Aufderheide, Sandro Steiner, Tobias Krebs, Bahman Asgharian, Richard Corley, Michael Oldham, Jason Adamson, Xiang Li, Irfan Rahman, Sonia Grego, Pei-Hsuan Chu, Shaun McCullough and Rodger Curren

In 2009, the passing of the Family Smoking Prevention and Tobacco Control Act facilitated the establishment of the FDA Center for Tobacco Products (CTP), and gave it regulatory authority over the marketing, manufacture and distribution of tobacco products, including those termed 'modified risk'. On 4–6 April 2016, the Institute for In Vitro Sciences, Inc. (IIVS) convened a workshop conference entitled, In Vitro Exposure Systems and Dosimetry Assessment Tools for Inhaled Tobacco Products, to bring together stakeholders representing regulatory agencies, academia and industry to address the research priorities articulated by the FDA CTP. Specific topics were covered to assess the status of current in vitro smoke and aerosol/vapour exposure systems, as well as the various approaches and challenges to quantifying the complex exposures in in vitro pulmonary models developed for evaluating adverse pulmonary events resulting from tobacco product exposures. The four core topics covered were: a) Tobacco Smoke and E-Cigarette Aerosols&#59; b) Air–Liquid Interface-In Vitro Exposure Systems&#59; c) Dosimetry Approaches for Particles and Vapours/In Vitro Dosimetry Determinations&#59; and d) Exposure Microenvironment/Physiology of Cells. The 2.5-day workshop included presentations from 20 expert speakers, poster sessions, networking discussions, and breakout sessions which identified key findings and provided recommendations to advance these technologies. Here, we will report on the proceedings, recommendations, and outcome of the April 2016 technical workshop, including paths forward for developing and validating non-animal test methods for tobacco product smoke and next generation tobacco product aerosol/vapour exposures. With the recent FDA publication of the final deeming rule for the governance of tobacco products, there is an unprecedented necessity to evaluate a very large number of tobacco-based products and ingredients. The questionable relevance, high cost, and ethical considerations for the use of in vivo testing methods highlight the necessity of robust in vitro approaches to elucidate tobacco-based exposures and how they may lead to pulmonary diseases that contribute to lung exposure-induced mortality worldwide.
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Assessment of In Vitro COPD Models for Tobacco Regulatory Science: Workshop Proceedings, Conclusions and Paths Forward for In Vitro Model Use

Holger Behrsing, Hans Raabe, Joseph Manuppello, Betsy Bombick, Rodger Curren, Kristie Sullivan, Sanjay Sethi, Richard Phipps, Yohannes Tesfaigzi, Sherwin Yan, Carl D’Ruiz, Robert Tarran, Samuel Constant, Gary Phillips, Marianna Gaça, Patrick Hayden, Xuefei Cao, Carole Mathis, Julia Hoeng, Armin Braun and Erin Hill

The Family Smoking Prevention and Tobacco Control Act of 2009 established the Food and Drug Administration Center for Tobacco Products (FDA-CTP), and gave it regulatory authority over the marketing, manufacture and distribution of tobacco products, including those termed 'modified risk'. On 8-10 December 2014, IIVS organised a workshop conference, entitled Assessment of In Vitro COPD Models for Tobacco Regulatory Science, to bring together stakeholders representing regulatory agencies, academia, industry and animal protection, to address the research priorities articulated by the FDA-CTP. Specific topics were covered to assess the status of current in vitro technologies as they are applied to understanding the adverse pulmonary events resulting from tobacco product exposure, and in particular, the progression of chronic obstructive pulmonary disease (COPD). The four topics covered were: a) Inflammation and Oxidative Stress; b) Ciliary Dysfunction and Ion Transport; c) Goblet Cell Hyperplasia and Mucus Production; and d) Parenchymal/Bronchial Tissue Destruction and Remodelling. The 2.5 day workshop included 18 expert speakers, plus poster sessions, networking and breakout sessions, which identified key findings and provided recommendations to advance the in vitro technologies and assays used to evaluate tobacco-induced disease etiologies. The workshop summary was reported at the 2015 Society of Toxicology Annual Meeting, and the recommendations led to an IIVS-organised technical workshop in June 2015, entitled Goblet Cell Hyperplasia, Mucus Production, and Ciliary Beating Assays, to assess these assays and to conduct a proof-of-principle multi-laboratory exercise to determine their suitability for standardisation. Here, we report on the proceedings, recommendations and outcomes of the December 2014 workshop, including paths forward to continue the development of non-animal methods to evaluate tissue responses that model the disease processes that may lead to COPD, a major cause of mortality worldwide.

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In Vitro Models for Studying Renal Stone Formation: A Clear Alternative

Felix Grases, Rafael M. Prieto and Antonia Costa-Bauzá

This paper discusses the limitations of using laboratory animals for direct in vivo observation of the development of renal stones. In fact, the majority of hypotheses related to mechanisms of stone formation have been based on the results of in vitro experiments. The relevance of in vitro experiments that allow the study of urolithiasis depends upon the degree of correspondence between the experimental conditions and those prevailing in the stone-forming kidney in vivo. For this reason, several in vitro experimental systems that attempt to reproduce the conditions found in vivo have been developed in order to study renal stone formation, which have been classified into two main groups: a) models to study papillary stone formation; and b) models to study “sedimentary” stone formation. These models are briefly described in this paper, and the information obtained was compared with that resulting from a study of the fine structure of real human renal calculi, in order to prove the validity of the models. It was concluded that the experimental in vitro models can closely reproduce the renal conditions under which human calculi are developed. This allows important data to be obtained about the aetiology of renal lithiasis, which is of great relevance to the development of effective treatments for this disease. Therefore, experimental in vitro models constitute a clear alternative to the use of laboratory animals.
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Biokinetic and Toxicodynamic Modelling and its Role in Toxicological Research and Risk Assessment

Bas J. Blaauboer

Toxicological risk assessment for chemicals is still mainly based on highly standardised protocols for animal experimentation and exposure assessment. However, developments in our knowledge of general physiology, in chemicobiological interactions and in (computer-supported) modelling, have resulted in a tremendous change in our understanding of the molecular mechanisms underlying the toxicity of chemicals. This permits the development of biologically based models, in which the biokinetics as well as the toxicodynamics of compounds can be described. In this paper, the possibilities are discussed of developing systems in which the systemic (acute and chronic) toxicities of chemicals can be quantified without the heavy reliance on animal experiments. By integrating data derived from different sources, predictions of toxicity can be made. Key elements in this integrated approach are the evaluation of chemical functionalities representing structural alerts for toxic actions, the construction of biokinetic models on the basis of non-animal data (for example, tissue–blood partition coefficients, in vitro biotransformation parameters), tests or batteries of tests for determining basal cytotoxicity, and more-specific tests for evaluating tissue or organ toxicity. It is concluded that this approach is a useful tool for various steps in toxicological hazard and risk assessment, especially for those forms of toxicity for which validated in vitro and other non-animal tests have already been developed.
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Neurotoxicology: Principles and Considerations of In Vitro Assessment

Michael Aschner and Tore Syversen

Neurotoxicology is an exciting area of science, not only because of the importance of toxic injury to the nervous system in human disease, but also because specific toxicants have served as invaluable tools for the advancement of our knowledge of “normal” neurobiological processes. In fact, much of our understanding of the organisation and function of the nervous system is based on observations derived from the actions of neurotoxicants. This paper addresses various physiological aspects behind the exquisite sensitivity of the nervous system to toxic agents, including the privileged status of the nervous system visà-vis blood-brain barrier function, the extensions of the nervous system over space and the requirements of cells with such a complex geometry, and the transmission of information across extracellular space. In addition, in vitro models and their utility in the assessment of neurotoxicological outcome are discussed, with reference to both their advantages and disadvantages.
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Organotypic Brain Slice Cultures: An Efficient and Reliable Method for Neurotoxicological Screening and Mechanistic Studies

Jens Noraberg

This paper reviews the current state of the use of organotypic brain slice cultures for neurotoxicological and neuropharmacological screening and mechanistic studies, as exemplified by excitotoxin application. At present, no in vitro systems have been approved by the regulatory authorities for neurotoxicity testing. For the evaluation of the slice culture method, organotypic hippocampal slice cultures were exposed to toxic doses of the excitotoxins, glutamate, N-methyl-D-aspartate (NMDA), kainic acid and 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and the glial toxin, DL-α-aminoadipic acid (DLAAA). Neuronal cell death was quantified by propidium iodide (PI) uptake, and visualised by Fluoro-Jade (FJ) staining. General cell death was monitored by lactate dehydrogenase (LDH) release into the culture medium. EC50 values for the different compounds, based on PI uptake after exposure for 48 hours in entire cultures, were: glutamate, 3.5mM; DL-AAA, 2.3mM; kainic acid, 13μM; NMDA, 11μM; and AMPA, 3.7μM. In the slice cultures, the hippocampal subfields displayed the same differences in vulnerability as those observed in vivo. When subfield analysis was performed on the cultures, the CA1 subfield was most susceptible to glutamate, NMDA and AMPA, while CA3 was most susceptible to kainic acid. The amount of LDH release for DL-AAA was about four times that of L-glutamate, in accordance with the additional toxic effect on glial cells, which was also found by confocal microscopy to stain for FJ. In conclusion, it was found that organotypic brain slice culture, combined with standardised protocols and quantifiable markers, such as PI and FJ staining, is a relevant and feasible in vitro system for neurotoxicity testing. Considering the amount and quality of the available published data, it is recommended that the brain slice culture method could be subjected to pre-validation and formal validation for inclusion in a tiered in vitro neurotoxicity testing scheme to supplement and replace conventional animal tests.
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