Sonawane A, Vadloori B, Poosala S
… +19 more, Kandarova H, Kulkarni M, Olayanju A, Dey T, Saxena U, Smirnova L, Kanda Y, Reddy J, Dravida S, Biswas S, Vinken M, Gettayacamin M, Ahluwalia A, Mondini F, Bhattacharya S, Kulkarni P, Jacobsen KR, Vangala S, Millás AL
The fact that animal models fail to replicate human disease faithfully is now being widely accepted by researchers across the globe. As a result, they are exploring the use of alternatives to animal models. The time has...The fact that animal models fail to replicate human disease faithfully is now being widely accepted by researchers across the globe. As a result, they are exploring the use of alternatives to animal models. The time has come to refine our experimental practices, reduce the numbers and eventually replace the animals used in research with human-derived and human-relevant 3-D disease models. Oncoseek Bio-Acasta Health, which is an innovative biotechnology start-up company based in Hyderabad and Vishakhapatnam, India, organises an annual International Conference on 3Rs Research and Progress. In 2021, this conference was on 'Advances in Research Animal Models and Cutting-Edge Research in Alternatives'. This annual conference is a platform that brings together eminent scientists and researchers from various parts of the world, to share recent advances from their research in the field of alternatives to animals including new approach methodologies, and to promote practices to help refine animal experiments where alternatives are not available. This report presents the proceedings of the conference, which was held in hybrid mode (i.e. virtual and in-person) in November 2021.
VEGAHUB (www.vegahub.eu) is a repository of freely available, downloadable tools based on computational toxicology methodologies. The main software tool available in VEGAHUB is VEGA QSAR software encoding more than 90 qu...VEGAHUB (www.vegahub.eu) is a repository of freely available, downloadable tools based on computational toxicology methodologies. The main software tool available in VEGAHUB is VEGA QSAR software encoding more than 90 quantitative structure-activity relationship (QSAR) models for tens of endpoints for human toxicology, ecotoxicology, environmental, physico-chemical and toxicokinetic properties. However, beyond VEGA QSAR, VEGAHUB offers several other tools. Here, we present these resources, the possibilities to fully exploit them and the ways in which to integrate results provided by different VEGAHUB tools. Read-across and weight-of-evidence represent a major advantage of VEGAHUB. Integration between hazard and exposure is provided within innovative tools, which are specific for well-defined scenarios, such as those for cosmetic products. Prioritisation can be achieved by integrating results from 48 models. Finally, we highlight how some tools may not only fit predefined endpoints but also could be applied to general problems and research applications in the QSAR field. A couple of examples are provided, in which a critical assessment of the predictions and the documentation associated with the prediction are considered, in order to properly assess the quality of the results. These results may be associated with different levels of uncertainty or even be conflicting.
Liver cell lines obtained from hepatomas, for example, HepG2 cells, are commonly used in drug toxicity studies. However, functional hepatocyte-like cells derived from mesenchymal stem cells (MSCs) could be a better optio...Liver cell lines obtained from hepatomas, for example, HepG2 cells, are commonly used in drug toxicity studies. However, functional hepatocyte-like cells derived from mesenchymal stem cells (MSCs) could be a better option for use in the study of drug metabolism and toxicity. Overdose of acetaminophen (APAP) and excess alcohol consumption are common causes of liver damage. The objective of the present study was to investigate the use of MSC-derived hepatocyte-like cells (MSCdH) in the assessment of drug-induced liver injury (by using APAP and ethanol), and to compare the toxic effects observed in the MSCdH with those exhibited by HepG2 cells. MSCs were isolated from umbilical cord and their functionality confirmed by their ability to differentiate into adipocytes, osteocytes and hepatocyte-like cells. It was shown that the MSCs successfully differentiated into hepatocyte-like cells, and these cells were further characterised by using various enzyme assays and by assessing albumin secretion and urea synthesis. Cytotoxicity was evaluated in the HepG2 and MSCdH after exposure to ethanol and APAP, with cell viability being determined by using the MTT assay. After exposure to ethanol and to APAP, cell viability decreased in a concentration-dependent manner for both types of hepatocytes. The respective EC values of ethanol-induced toxicity for HepG2 and MSCdH cells were 2.5% and 1.3% v/v ( < 0.001); for APAP-induced toxicity they were 19.1 mM and 12.6 mM ( < 0.001). These findings show that there is a distinct difference between the two types of hepatocytes in terms of APAP-induced and ethanol-induced liver injury.
The precise determination of the intracellular concentration of a drug is a major challenge in drug discovery. Microinjection is a very effective technique for the introduction of macromolecules into single cells. Howeve...The precise determination of the intracellular concentration of a drug is a major challenge in drug discovery. Microinjection is a very effective technique for the introduction of macromolecules into single cells. However, due to the large number of parameters that need to be adjusted and the complex physical mechanisms involved, there are currently no means by which the concentration of a microinjected intracellular compound could be theoretically estimated. In this paper, we present a method for the theoretical estimation of intracellular drug concentration, based on the framework of classical fluid mechanism theory - specifically, the modified Bernoulli equation. We introduce into Bernoulli's classical equation the effect of friction due to the non-laminar regimes of the injected fluid. We also study the compatibility of our theoretical estimation model with variations in injection time and concentration of the compound inside the microinjection needle. Finally, microinjected calcium concentrations estimated with the theoretical model were compared with those determined experimentally in several cell types, by using a Fura-2-based Ca imaging technique.
The significance of contributions in volumes 11-46 (1983 to 2018) of in relation to the reduction, refinement and replacement of animal experimentation in biomedical research and testing is reviewed and discussed by the...The significance of contributions in volumes 11-46 (1983 to 2018) of in relation to the reduction, refinement and replacement of animal experimentation in biomedical research and testing is reviewed and discussed by the journal's former editor-in-chief, with particular emphasis on the development and production of the journal itself, FRAME, the European Centre for the Validation of Alternative Methods and other organisations. The role of the journal in promoting the principles of humane research (as spelled out by William Russell and Rex Burch) and highlighting a range of important issues and focus topics is explored. These include: botulinum toxin potency testing; ethical issues; the use of human volunteers, and human cells and tissues; the use of non-human primates (especially chimpanzees) and dogs as laboratory animals; toxicity testing in relation to cosmetics, pharmaceuticals and chemicals; UK and EU politics and legislation; and test validation and invalidation. The review concludes by identifying some of the issues that still need to be discussed and some of the questions that urgently need to be addressed.
Neural retinal organ cultures are used to investigate ocular pathomechanisms. However, these cultures lack the essential retinal pigment epithelium (RPE) cells, which are part of the actual retina. To simulate a more re...Neural retinal organ cultures are used to investigate ocular pathomechanisms. However, these cultures lack the essential retinal pigment epithelium (RPE) cells, which are part of the actual retina. To simulate a more realistic model, porcine neural retina explants were cocultured with ARPE-19 cells (ARPE-19 group), which are derived from human RPE. To identify whether the entire cells or just the cell factors are necessary, in a second experimental group, porcine neural retina explants were cultured with medium derived from ARPE-19 cells (medium group). Individually cultured neural retina explants served as controls (control group). After 8 days, all neural retinas were analysed to evaluate retinal thickness, photoreceptors, microglia, complement factors and synapses ( = 6-8 per group). The neural retina thickness in the ARPE-19 group was significantly better preserved than in the control group ( = 0.031). Also, the number of L-cones was higher in the ARPE-19 group, as compared to the control group ( < 0.001). Furthermore, the ARPE-19 group displayed an increased presynaptic glutamate uptake (determined via vGluT1 labelling) and enhanced post-synaptic density (determined via PSD-95 labelling). Combined Iba1 and iNOS detection revealed only minor effects of ARPE-19 cells on microglial activity, with a slight downregulation of total microglia activity apparent in the medium group. Likewise, only minor beneficial effects on photoreceptors and synaptic structure were found in the medium group. This novel system offers the opportunity to investigate interactions between the neural retina and RPE cells, and suggests that the inclusion of a RPE feeder layer has beneficial effects on the maintenance of neural retina. By modifying the culture conditions, this coculture model allows a better understanding of photoreceptor death and photoreceptor-RPE cell interactions in retinal diseases.
Prediction of chemical toxicity is very useful in risk assessment. With the current paradigm shift towards the use of and systems, we present herein a theoretical mathematical description of a quasi-diffusion process t...Prediction of chemical toxicity is very useful in risk assessment. With the current paradigm shift towards the use of and systems, we present herein a theoretical mathematical description of a quasi-diffusion process to predict chemical concentrations in 3-D spheroid cell cultures. By extending a 2-D Virtual Cell Based Assay (VCBA) model into a 3-D spheroid cell model, we assume that cells are arranged in a series of concentric layers within the sphere. We formulate the chemical quasi-diffusion process by simplifying the spheroid with respect to the number of cells in each layer. The system was calibrated and tested with acetaminophen (APAP). Simulated predictions of APAP toxicity were compared with empirical data from measurements by using a 3-D spheroid model. The results of this first attempt to extend the VCBA model are promising - they show that the VCBA model simulates close correlation between the influence of compound concentration and the viability of the HepaRG 3-D cell culture. The 3-D VCBA model provides a complement to current procedures to refine experimental setups, to fill data gaps and help in the interpretation of data for the purposes of risk assessment.
Since the inaugural issue of , many changes within publishing have occurred, impacting when, where, and how researchers conduct literature searches for non-animal alternatives. Such changes include increased rate of grow...Since the inaugural issue of , many changes within publishing have occurred, impacting when, where, and how researchers conduct literature searches for non-animal alternatives. Such changes include increased rate of growth in scientific publications, greater number of databases and online resources available to search, opportunities for open and almost immediate dissemination of research outputs such as preprints and method protocols, and the development of reporting guidelines for animal research. Here we offer a librarian's perspective on these changes and advice on how to manage them to enable robust and diverse alternatives to be implemented in future research.
testing has been the gold standard for preclinical drug development and toxicology. However, animal-based methods often lack human relevance and have a low predictability rate, not to mention the enormous ethical and fin...testing has been the gold standard for preclinical drug development and toxicology. However, animal-based methods often lack human relevance and have a low predictability rate, not to mention the enormous ethical and financial concerns associated with their use. For instance, according to the US Congressional Budget Office (cbo.gov), it takes an average of 10.5 years to take a compound from the preclinical phase to the market, with a cost of US$1-US$2 billion. 90% of drugs that are tested in animals and enter clinical trials fail due to lack of safety and efficacy - this fact questions the significance of testing. Cells in culture can recapitulate certain aspects of physiology and disease, as well as indicate drug responses and toxicity. Thus, they represent a sophisticated human-relevant and humane alternative. With advances in the field, it is anticipated that confidence will be gained towards a move away from traditional models. Specialised supplier industries have been a driving force in the transition to non-animal research, by translating new approach methodologies into scalable products that have been adopted by the regulatory and testing industries - but we are still at the beginning. In this article, we introduce the perspective of the device supplier industry on the current challenges and opportunities surrounding the adoption of new methods, with the goal of promoting effective co-operation with scientists and other stakeholders. In addition, we highlight some examples of where non-animal approaches have been used in regulatory submissions, as well as listing some educational and training resources that can help when selecting the most appropriate assay.
The need to reduce, refine and replace animal experimentation has led to a boom in the establishment of new approach methodologies (NAMs). This promising trend brings the hope that the replacement of animals by using NAM...The need to reduce, refine and replace animal experimentation has led to a boom in the establishment of new approach methodologies (NAMs). This promising trend brings the hope that the replacement of animals by using NAMs will become increasingly accepted by regulators, included in legislation, and consequently more-often implemented by industry. The majority of NAMs, however, are still not very well understood, either due to the complexity of the applied approach or the data analysis workflow. A potential solution to this problem is the provision of better educational resources to scientists new to the area - showcasing the added value of NAMs and outlining various ways of overcoming issues associated with knowledge gaps. In this paper, the educational exchange between four institutions - namely, two universities and two SMEs - via a series of video training sessions, is described. The goal of this exchange was to showcase an exemplary event to help introduce scientists to non-animal approaches, and to actively support the development of resources enabling the use of alternatives to laboratory animals.