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This study describes a complex human in vitro model for evaluating anti‐inflammatory drug response in the alveoli that may contribute to the reduction of animal testing in the pre‐clinical stage of drug development. The model is based on the human alveolar epithelial cell line Arlo co‐cultured with macrophages differentiated from the THP‐1 cell line, creating a physiological biological microenvironment. To mimic the three‐dimensional architecture and dynamic expansion and relaxation of the air‐blood‐barrier, they are grown on a stretchable microphysiological lung‐on‐chip. For validating the in vitro model, three different protocols have been developed to demonstrate the clinically established anti‐inflammatory effect of glucocorticoids to reduce certain inflammatory markers after different pro‐inflammatory stimuli: (1) an inflammation caused by bacterial LPS (lipopolysaccharides) to simulate an LPS‐induced acute lung injury measured best with cytokine IL‐6 release; (2) an inflammation caused by LPS at ALI (air‐liquid interface) to investigate aerosolized anti‐inflammatory treatment, measured with chemokine IL‐8 release; and (3) an inflammation with a combination of human inflammatory cytokines TNFα and IFNγ to simulate a critical cytokine storm leading to epithelial barrier disruption, where the eventual weakening or protection of the epithelial barrier can be measured. In all cases, the presence of macrophages appeared to be crucial to mediating inflammatory changes in the alveolar epithelium. LPS induction led to inflammatory changes independently of stretch conditions. Dynamic stretch, emulating breathing‐like mechanics, was essential for in vitro modeling of the clinically relevant outcome of epithelial barrier disruption upon TNFα/IFNγ‐induced inflammation. [ABSTRACT FROM AUTHOR]

While neurosurgical interventions are frequently used in laboratory mice, refinement efforts to optimize analgesic management based on multimodal approaches appear to be rather limited. Therefore, we compared the efficacy and tolerability of combinations of the non-steroidal anti-inflammatory drug carprofen, a sustained-release formulation of the opioid buprenorphine, and the local anesthetic bupivacaine with carprofen monotherapy. Female and male C57BL/6J mice were subjected to isoflurane anesthesia and an intracranial electrode implant procedure. Given the multidimensional nature of postsurgical pain and distress, various physiological, behavioral, and biochemical parameters were applied for their assessment. The analysis revealed alterations in Neuro scores, home cage locomotion, body weight, nest building, mouse grimace scales, and fecal corticosterone metabolites. A composite measure scheme allowed the allocation of individual mice to severity classes. The comparison between groups failed to indicate the superiority of multimodal regimens over high-dose NSAID monotherapy. In conclusion, our findings confirmed the informative value of various parameters for assessment of pain and distress following neurosurgical procedures in mice. While all drug regimens were well tolerated in control mice, our data suggest that the total drug load should be carefully considered for perioperative management. Future studies would be of interest to assess potential synergies of drug combinations with lower doses of carprofen. [ABSTRACT FROM AUTHOR]

The EU adopted Directive 2010/63/EU, thereby creating an instrument for legally implementing effective measures to regulate animal experimentation. In this framework, the 3Rs principle for replacement, reduction, and refinement of animal experiments proposed by Russell and Burch (1959) is of paramount importance. The highest goal of the 3Rs can be understood as a Culture of Care (CoC), entailing complete renunciation of sentient beings as test subjects. The implementation of a CoC is explicitly mentioned in Recital 31 of the EU-Directive. This article elaborates on historical underpinnings of the CoC concept and uses qualitative social research to trace its characteristics. Data were based on non-standardized survey procedures (topic-oriented, guideline-based expert interviews) and were collected at the management, scientific, supervisory and care levels in relation to animal experimentation. The goal of the qualitative approach is to understand contexts, conditions, strategies, and consequences of the CoC and depict them as a theoretical model. [ABSTRACT FROM AUTHOR]

Copyright of Environmental & Social Management Journal / Revista de Gestão Social e Ambiental is the property of Environmental & Social Management Journal and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Abstract Site-specific recombinases (SSRs) are critical for achieving precise spatiotemporal control of engineered alleles. These enzymes play a key role in facilitating the deletion or inversion of loci flanked by recombination sites, resulting in the activation or repression of endogenous genes, selection markers or reporter elements. However, multiple recombination in complex alleles can be laborious. To address this, a new and efficient method using AAV vectors has been developed to simplify the conversion of systems based on Cre, FLP, Dre and Vika recombinases. In this study, we present an effective method for ex vivo allele conversion using Cre, FLP (flippase), Dre, and Vika recombinases, employing adeno-associated viruses (AAV) as delivery vectors. AAVs enable efficient allele conversion with minimal toxicity in a reporter mouse line. Moreover, AAVs facilitate sequential allele conversion, essential for fully converting alleles with multiple recombination sites, typically found in conditional knockout mouse models. While simple allele conversions show a 100% efficiency rate, complex multiple conversions consistently achieve an 80% conversion rate. Overall, this strategy markedly reduces the need for animals and significantly speeds up the process of allele conversion, representing a significant improvement in genome engineering techniques.

Abstract While neurosurgical interventions are frequently used in laboratory mice, refinement efforts to optimize analgesic management based on multimodal approaches appear to be rather limited. Therefore, we compared the efficacy and tolerability of combinations of the non-steroidal anti-inflammatory drug carprofen, a sustained-release formulation of the opioid buprenorphine, and the local anesthetic bupivacaine with carprofen monotherapy. Female and male C57BL/6J mice were subjected to isoflurane anesthesia and an intracranial electrode implant procedure. Given the multidimensional nature of postsurgical pain and distress, various physiological, behavioral, and biochemical parameters were applied for their assessment. The analysis revealed alterations in Neuro scores, home cage locomotion, body weight, nest building, mouse grimace scales, and fecal corticosterone metabolites. A composite measure scheme allowed the allocation of individual mice to severity classes. The comparison between groups failed to indicate the superiority of multimodal regimens over high-dose NSAID monotherapy. In conclusion, our findings confirmed the informative value of various parameters for assessment of pain and distress following neurosurgical procedures in mice. While all drug regimens were well tolerated in control mice, our data suggest that the total drug load should be carefully considered for perioperative management. Future studies would be of interest to assess potential synergies of drug combinations with lower doses of carprofen.

Introduction: Veterinary vaccines against Clostridium perfringens type C need to be tested for absence of toxicity, as mandated by pharmacopoeias worldwide. This toxicity testing is required at multiple manufacturing steps and relies on outdated mouse tests that involve severe animal suffering. Clostridium perfringens type C produces several toxins of which the b-toxin is the primary component responsible for causing disease. Here, we describe the successful development of a new cell-based in vitro assay that can address the specific toxicity of the b-toxin. Methods: Development of the cell-based assay followed the principle of in vitro testing developed for Cl. septicum vaccines, which is based on Vero cells. We screened four cell lines and selected the THP-1 cell line, which was shown to be the most specific and sensitive for b-toxin activity, in combination with a commercially available method to determine cell viability (MTS assay) as a readout. Results: The current animal test is estimated to detect 100 - 1000-fold dilutions of the Cl. perfringens type C non-inactivated antigen. When tested with an active Cl. perfringens type C antigen preparation, derived from a commercial vaccine manufacturing process, our THP-1 cell-based assay was able to detect toxin activity from undiluted to over 10000-fold dilution, showing a linear range between approximately 1000- and 10000-fold dilutions. Assay specificity for the b-toxin was confirmed with neutralizing antibodies and lack of reaction to Cl. perfringens culture medium. In addition, assay parameters demonstrated good repeatability. Conclusions: Here, we have shown proof of concept for a THP-1 cell-based assay for toxicity testing of veterinary Cl. perfringens type C vaccines that is suitable for all vaccine production steps. This result represents a significant step towards the replacement of animal-based toxicity testing of this veterinary clostridial antigen. As a next step, assessment of the assay's sensitivity and repeatability and validation of the method will have to be performed in a commercial manufacturing context in order to formally implement the assay in vaccine quality control. [ABSTRACT FROM AUTHOR]

Background: Colorectal cancer (CRC) is one of the most common and deadly malignant neoplasms worldwide. It is associated with multiple lifestyle risk factors such as poor diet, physical inactivity, alcohol and red meat consumption, smoking, and obesity. Consequently, studies have been conducted on the protective activity of bioactive compounds present in some foods against CRC, making it necessary to understand how diet can affect human health. In this sense, coffee, the second most consumed beverage in the world, is a major source of phytochemicals associated with a possible antitumor effect on CRC cells. Purpose: This study aimed to analyze the chemopreventive potential of green and roasted coffee extracts on HT-29 CRC cells cultured in 2D and 3D models. Methods: Flow cytometry was employed to quantify propidium iodide and DiOC6 uptake, ROS production, phosphatidylserine exposure, and cell cycle progression. In addition, cells and spheroids were analyzed by light microscopy to identify morphological changes. Results: The results showed that, under the employed conditions, treatment with both extracts had a concentration-dependent cytotoxic effect. Decreased cell viability was associated with mitochondrial membrane hyperpolarization, prooxidant effect, increased phosphatidylserine exposure, and changes in the G1 phase of the cell cycle. Conclusion: For the evaluated cell markers, 3D cultures required higher doses to achieve significant cytotoxic effects when compared to monolayer cultures. Since 3D cultures favor the formation of complex cell–cell interactions, this model offers a closer look at in vivo conditions for evaluating the toxicity and preclinical efficacy of potential chemopreventive agents for CRC. The experimental evidence from this study supports the 3R principles of replacement, reduction, and refinement for toxicological research in the pharmaceutical industry.

Simple Summary: Scientific experiments that use animals are strictly regulated, mainly as a result of public concerns about animal testing. Examination of the regulation of animal experiments shows that most of the experiments submitted for authorization are approved. But what could be the reasons for this high approval? Since public understanding is needed in order to reach a common agreement that will shape animal research policy, it is important to have a transparent discussion about what leads to the frequent approval of animal experiments. The aim of this article is to discuss why frequent approval of experiments is a problem: the regulatory process has been put in place to authorize only acceptable experiments. Even though some experiments, such as cosmetic testing on animals, are prohibited in many countries irrespective of harm–benefit ratios, most types of animal experiments are approved. Further, we explain some possible genuine reasons for frequent approval, but at the same time, we also discuss how the regulatory process could be leading to the approval of unacceptable studies. To ensure transparency, gain public trust, and improve the understanding of the regulation of animal testing, we propose that regulators publish their experiment evaluations on an accessible online platform, explaining how they reached their decisions. The use of nonhuman animals in biomedical research is regulated under stringent conditions, not only in response to societal attitudes towards animal experimentation but also because ethical responsibility in scientific research requires researchers and veterinarians to be more invested and aim to improve the welfare of animals used for experiments. Analyses of animal research oversight reveal the frequent approval of experiments, and the approval of some experiments has raised and continues to raise public concerns. Societal compliance is required for a consensus-based approach to animal research policy, prompting the need to have transparent discussions about oversight and the frequency of approvals. We discuss how frequent approval may be perceived and why it seems problematic from a societal perspective: the regulatory process exists to approve only legitimate experiments. Although some experiments remain unacceptable irrespective of their harm–benefit ratios, almost all experiments are approved. We explain some possible legitimate reasons for frequent approval and how the review process could be leading to the approval of illegitimate studies. To ensure transparency and improve public trust and understanding of oversight, we propose the adoption of a platform to inform society about how unethical experiments are screened out. [ABSTRACT FROM AUTHOR]

In the healthcare system, lower leg fractures remain relevant, incurring costs related to surgical treatment, hospitalization, and rehabilitation. The duration of treatment may vary depending on the individual case and its severity. Casting as a post-surgical fracture treatment is a common method in human and experimental veterinary medicine. Despite the high importance of sheep in preclinical testing materials for osteosynthesis, there is no standardised cast system ensuring proper stabilisation and functionality of hind limbs during the healing of tibia fractures or defects. Existing treatment approaches for tibial osteosynthesis in laboratory animal science include sling hanging, external fixators, or former Achilles tendon incision. These methods restrict animal movement for 4–6 weeks, limit species-typical behaviour, and impact social interactions. Our pilot study introduces a Standardised Walking Cast (SWC) for sheep, enabling immediate physiological movement post surgery. Seven Rhone sheep (female, 63.5 kg ± 6.45 kg) each with a single tibia defect (6 mm mechanical drilled defect) underwent SWC application for 4 weeks after plate osteosynthesis. The animals bore weight on their operated leg from day one, exhibiting slight lameness (grade 1–2 out of 5). Individual step lengths showed good uniformity (average deviation: 0.89 cm). Group housing successfully started on day three after surgery. Weekly X-rays and cast changes ensured proper placement, depicting the healing process. This study demonstrates the feasibility of using an SWC for up to 72 kg of body weight without sling hanging via ceiling mounting or external fixation techniques. Allowing species-typical movement and social behaviour can significantly improve the physiological behaviour of sheep in experiments, contributing to refinement. [ABSTRACT FROM AUTHOR]

To establish an ethical, reliable, and expandable retinal pigment epithelial (RPE) cell model with maintained RPE properties compatible with multifarious assays. RPE cells from abattoir-obtained porcine eyes were cultured under various conditions. Morphology, RPE cell-specific protein markers (RPE-65, CRALBP), and the tight junction marker ZO-1 were analyzed by phase-contrast microscopy, immunocytochemistry, and western blot, and transepithelial electrical resistance (TEER) was determined to assess barrier function. The porcine RPE cells (pRPE) were best established using TrypLE Express, 10% fetal bovine serum (FBS) supplemented high-glucose media, and subculturing at semi-confluency. The pRPE cells maintained epithelioid morphology with ZO-1 positive tight junctions at the cell-to-cell borders, the ability to establish proper barrier function (TEERmax: 346/375 Ω⋅cm2 at passage I/passage VI), and expressed CRALBP and RPE-65 for several passages. The RPE characteristics decreased and disappeared with transdifferentiation. This work describes, for the first time, a pRPE cell model that exhibits preserved RPE properties for several passages on cell culture plastic plates. Though RPE characteristics were maintained for at least 6 passages, the reduced CRALBP and RPE-65 with passaging emphasize that lower passage cells are advantageous to utilize, and that morphology, barrier function, and ZO-1 localization cannot be solely employed as a quality measure of RPE identity. Pigs are phylogenetically similar to humans, including similar physiology, anatomy and immune system. Therefore, porcine RPE cells constitute a relevant model system for studying human eye diseases, such as AMD. [ABSTRACT FROM AUTHOR]

Developed countries have adopted the circular economy concept to solve environmental problems. This study underpins the paradox theory to investigate the circular economy's implementation status in the ASEAN region through the circular economy index. The circular economy index disclosure data for top companies in the ASEAN region was collected through an automated content analysis approach using DICTION on the annual/sustainability reports. The findings show that most public companies from the ASEAN region disclose the circular economy indicators in their annual/sustainability reports. The environment-sensitive firms are disclosing the circular economy indicators actively compared to the non-environment sensitive companies. Based on the results, this study provides theoretical and practical implications. This study is also limited because it is based only on the ASEAN region. Future studies can underpin other theoretical support and provide a comparative analysis across the continents. [ABSTRACT FROM AUTHOR]

Since the start of the COVID‐19 pandemic, a tremendous amount of work has been done by scientists to understand the pathogenesis of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Several animal models have been reported to be useful for the development and testing of vaccines, antivirals, or therapeutics. These in vivo experiments have to be performed at high containment biosafety level (BSL)-3. Thus, an appropriate biosafety, facility and data management as well as high standard of animal welfare, thorough study design, and staff training combined with an overall research transparency and open communication need to be implemented to achieve high quality reproducible research. Such an approach combined with the well-known 3R principle (Replacement, Reduction, and Refinement) and the 3S principle (Good Science, Good Sense, and Good Sensibilities) can contribute to culture of care in scientific research. The perspective reviews the key points of optimized biosafety, data, quality and animal welfare management in high risk biosafety-settings to conduct animal experiments with infectious pathogens in the context of a culture of care. [ABSTRACT FROM AUTHOR]

Salah satu masalah lingkungan yang mempengaruhi kesehatan lingkungan dan manusia adalah sampah yang dihasilkan dari berbagai aktivitas manusia. Mengelola serta memilah sampah berdasarkan jenisnya akan mempermudah pemilihan langkah pemrosesan berikutnya, yang akan membantu menguranginya. Sekolah SMA AL-HUDA Cengkareng merupakan salah satu satuan pendidikan dengan jenjang sekolah menengah atas di Cengkareng, Jakarta Barat yang memiliki siswa cukup banyak. Sekolah ini dalam kegiatannya di bawah Kementrian Pendidikan dan Kebudayaan. Tujuan dari kegiatan ini adalah untuk meningkatkan pemahaman siswa tentang pengelolaan dan pemilahan sampah. Metode yang digunakan adalah dengan memberikan materi dan praktek langsung tata cara pengolahan kompos Takakura Home Method, membagi siswa menjadi kelompok-kelompok kecil serta memberikan praktek langsung cara pembuatan dan pengolahan kompos Takakura Home Method. Hasil dari kegiatan ini siswa memahami informasi tentang jenis sampah, pengertiannya, efeknya, jenisnya, dan pengelolaannya sehingga menjadi bahan yang berguna.

Background: Effective communication is crucial for broad acceptance and applicability of alternative methods in 3R biomedical research and preclinical testing. 3D bioprinting is used to construct intricate biological structures towards functional liver models, specifically engineered for deployment as alternative models in drug screening, toxicological investigations, and tissue engineering. Despite a growing number of reviews in this emerging field, a comprehensive study, systematically assessing practices and reporting quality for bioprinted liver models is missing. Methods: In this systematic scoping review we systematically searched MEDLINE (Ovid), EMBASE (Ovid) and BioRxiv for studies published prior to June 2nd, 2022. We extracted data on methodological conduct, applied bioinks, the composition of the printed model, performed experiments and model applications. Records were screened for eligibility and data were extracted from included articles by two independent reviewers from a panel of seven domain experts specializing in bioprinting and liver biology. We used RAYYAN for the screening process and SyRF for data extraction. We used R for data analysis, and R and Graphpad PRISM for visualization. Results: Through our systematic database search we identified 1042 records, from which 63 met the eligibility criteria for inclusion in this systematic scoping review. Our findings revealed that extrusion-based printing, in conjunction with bioinks composed of natural components, emerged as the predominant printing technique in the bioprinting of liver models. Notably, the HepG2 hepatoma cell line was the most frequently employed liver cell type, despite acknowledged limitations. Furthermore, 51% of the printed models featured co-cultures with non-parenchymal cells to enhance their complexity. The included studies offered a variety of techniques for characterizing these liver models, with their primary application predominantly focused on toxicity testing. Among the frequently analyzed liver markers, albumin and urea stood out. Additionally, Cytochrome P450 (CYP) isoforms, primarily CYP3A and CYP1A, were assessed, and select studies employed nuclear receptor agonists to induce CYP activity. Conclusion: Our systematic scoping review offers an evidence-based overview and evaluation of the current state of research on bioprinted liver models, representing a promising and innovative technology for creating alternative organ models. We conducted a thorough examination of both the methodological and technical facets of model development and scrutinized the reporting quality within the realm of bioprinted liver models. This systematic scoping review can serve as a valuable template for systematically evaluating the progress of organ model development in various other domains. The transparently derived evidence presented here can provide essential support to the research community, facilitating the adaptation of technological advancements, the establishment of standards, and the enhancement of model robustness. This is particularly crucial as we work toward the long-term objective of establishing new approach methods as reliable alternatives to animal testing, with extensive and versatile applications.

IntroductionVeterinary vaccines against Clostridium perfringens type C need to be tested for absence of toxicity, as mandated by pharmacopoeias worldwide. This toxicity testing is required at multiple manufacturing steps and relies on outdated mouse tests that involve severe animal suffering. Clostridium perfringens type C produces several toxins of which the β-toxin is the primary component responsible for causing disease. Here, we describe the successful development of a new cell-based in vitro assay that can address the specific toxicity of the β-toxin.MethodsDevelopment of the cell-based assay followed the principle of in vitro testing developed for Cl. septicum vaccines, which is based on Vero cells. We screened four cell lines and selected the THP-1 cell line, which was shown to be the most specific and sensitive for β-toxin activity, in combination with a commercially available method to determine cell viability (MTS assay) as a readout. ResultsThe current animal test is estimated to detect 100 – 1000-fold dilutions of the Cl. perfringens type C non-inactivated antigen. When tested with an active Cl. perfringens type C antigen preparation, derived from a commercial vaccine manufacturing process, our THP-1 cell-based assay was able to detect toxin activity from undiluted to over 10000-fold dilution, showing a linear range between approximately 1000- and 10000-fold dilutions. Assay specificity for the β-toxin was confirmed with neutralizing antibodies and lack of reaction to Cl. perfringens culture medium. In addition, assay parameters demonstrated good repeatability.ConclusionsHere, we have shown proof of concept for a THP-1 cell-based assay for toxicity testing of veterinary Cl. perfringens type C vaccines that is suitable for all vaccine production steps. This result represents a significant step towards the replacement of animal-based toxicity testing of this veterinary clostridial antigen. As a next step, assessment of the assay’s sensitivity and repeatability and validation of the method will have to be performed in a commercial manufacturing context in order to formally implement the assay in vaccine quality control.

Introduction There are rational arguments to replace existing in vivo potency and safety assays for batch release testing of vaccines with more advanced non-animal techniques to measure critical quality attributes. However, the introduction of in vitro alternatives to replace in vivo release assays of authorized vaccines is challenging. Areas covered This report describes the hurdles encountered in substituting in vivo assays and ways to overcome these and provides arguments why more advanced in vitro alternatives are superior, not only as a tool to monitor the quality of vaccines but also from a practical, economical, and ethical point of view. The rational arguments provided for regulatory acceptance can support a strategy to replace/substitute any in vivo batch release test if an appropriate non-animal testing strategy is available. Expert opinion For several vaccines, in vivo release assays have been replaced leading to an optimized control strategy. For other vaccines, new assays are being developed that can expect to be introduced within 5–10 years. From a scientific, logistical, and animal welfare perspective, it would be beneficial to substitute all existing in vivo batch release assays for vaccines. Given the challenges related to development, validation, and acceptance of new methods, and considering the relatively low prices of some legacy vaccines, this cannot be done without government incentives and supportive regulatory authorities from all regions.

In mammalian research, the implementation of the 3Rs is ubiquitous. However, the adaptation of procedures for experimental work on fish seems less extensive in comparison, even though fish model organisms are common in a broad range of research fields already. To strengthen animal welfare in experimental research, we provide an overview of current research results, including studies on the nociception of fishes. Furthermore, we describe the potential of implementing the 3Rs in fish experimental research. In the context of "Reduction", we show alternative research methods to lethal sampling. Considering "Refinement", we point out possibilities to improve fish handling and indicate that adaptations to the individual species ecology are necessary. Under the aspect of "Replacement", we describe the high potential of cell cultures that can be obtained from fish tissue and give an overview of the already extensive use in ecotoxicology and virology. In addition, we illustrate that cell cultures could also be increasingly used for basic research. [ABSTRACT FROM AUTHOR]

The pharmacokinetic (PK) and toxicokinetic profile of a drug from its preclinical evaluation helps the researcher determine whether the drug should be tested in humans based on its safety and toxicity. Preclinical studies require time and resources and are prone to error. Moreover, according to the United States Food and Drug Administration Modernisation Act 2, animal testing is no longer mandatory for new drug development, and an animal-free alternative, such as cell-based assay and computer models, can be used. Different physiologically based PK models were developed for an anaplastic lymphoma kinase inhibitor in rats and monkeys after intravenous and oral administration using its physicochemical properties and in vitro characterisation data. The developed model was validated against the in vivo data available in the literature, and the validation results were found within the acceptable limit. A parameter sensitivity analysis was performed to identify the properties of the compound influencing the PK profile. This work demonstrates the application of the physiologically based PK model to predict the PKs of a drug, which will eventually assist in reducing the number of animal studies and save time and cost of drug discovery and development. [ABSTRACT FROM AUTHOR]

Copyright of Rare Metals is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Life cycle strategies for traction batteries, such as remanufacturing, reuse, and recycling of retired automotive lithium-ion batteries (LIBs), have received growing attention, as large volumes of LIBs will retire in the near future and the demand for LIBs continues to grow. At the same time, the relevance of the sustainability of a battery system over its entire life cycle is increasing as factors such as the EU Battery Regulation provide greater market and product transparency. As a result, research and industry require forecasts in order to assess the future market situation and to make well-founded decisions. Therefore, this paper provides forecasts of the return volumes of battery systems from BEVs and PHEVs up to 2035. Additionally, a representative European battery pack for PHEVs and BEVs was evaluated for each year since 2013, based on the ten vehicles with the largest market share in each year until 2021. In addition, the battery return streams are divided into three different 3R strategies based on expert interviews in order to evaluate the upcoming workload in these areas. The term "3R" refers to the sum of the currently existing pathways around reuse, remanufacturing, and recycling. In 2030, about 38.8 GWh will return and enter the recycling process annually. For battery reuse, about 13 GWh will return every year from 2030 onwards, ready to be used in stationary storage for energy transition. Compared to this, battery remanufacturing is expected to be supplied with a slightly lower volume of approximately 11 GWh per year. [ABSTRACT FROM AUTHOR]

Abstract The use of novel tools and technologies is transforming our approaches to study wild animal behaviour. Yet, from disturbance of natural habitats through field experiments to tagging, there is much ground for researchers to provide clear ethical assessment of their research practices and how they can affect wild animals. In this perspective, I review the current trends in field animal behaviour research, with a focus on studies on sociality, and investigate the forms under which such practices can impact animals. I focus particularly on the notions of disturbance and invasiveness. I build a parallel with the legal classifications in terms of degrees of severity found in the Swiss law and aimed at protecting animal well‐being, inspired by the principles of the 3R (Replace, Refine, Reduce) used in invasive animal research. I highlight how the impact of research can be assessed and monitored, particularly through behavioural and physiological means, identifying areas where such monitoring is critical, but also acknowledging the theoretical and practical limitations to such tools. I hope to trigger discussions on an ethical use of new tools and technologies, which can both inspire new research while limiting the disturbance to wild animals.

Simple Summary: The recent United States Pharmacopeia general chapter <121> requires a non-quantitative bioidentity test either as a rabbit blood sugar assay or as an in vitro insulin cell-based assay using in-cell Western (ICW) technology for insulin batch release. However, for quantification during stability or comparability studies, the rabbit blood sugar test is still required using a minimum of 24 rabbits to obtain one result. Based on the 3R principle (replace, reduce, and refine), this study sought to qualify the in vitro ICW cell-based bioassay approach for quantifying insulin activity. A bridging study with different insulins and stress samples revealed a clear correlation between the in vitro and in vivo test results. The replacement of the animal-based assay with the quantitative in vitro ICW cell-based bioassay for batch quality control saved cost, reduced cycle times while obtaining more meaningful and reliable data, and, above all else, reduced the suffering of many rabbits. For the quantification of insulin activity, United States Pharmacopeia (USP) general chapter <121> continues to require the rabbit blood sugar test. For new insulin or insulin analogue compounds, those quantitative data are expected for stability or comparability studies. At Sanofi, many rabbits were used to fulfil the authority's requirements to obtain quantitative insulin bioactivity data until the in vivo test was replaced. In order to demonstrate comparability between the in vivo and in vitro test systems, this study was designed to demonstrate equivalency. The measurement of insulin lispro and insulin glargine drug substance and drug product batches, including stress samples (diluted or after temperature stress of 30 min at 80 °C), revealed a clear correlation between the in vitro and in vivo test results. The recovery of quantitative in vitro in-cell Western (ICW) results compared to the in vivo test results was within the predefined acceptance limits of 80% to 125%. Thus, the in vitro ICW cell-based bioassay leads to results that are equivalent to the rabbit blood sugar test per USP <121>, and it is highly suitable for insulin activity quantification. For future development compounds, the in vitro in-cell Western cell-based assay can replace the rabbit blood sugar test required by USP <121>. [ABSTRACT FROM AUTHOR]

Simple Summary: Blood sampling from laboratory animals is a routine procedure in biomedical research, and husbandry is known to affect the quality of animal models. The effect of handling required by these techniques can have a major impact on the condition and responses of experimental animals. Comparing three common methods of blood extraction in mice, i.e., saphenous vein phlebotomy, caudal vein phlebotomy and tail cut blood collection, together with the effect of acclimation, we study which technique is less stressful for the animal. Our results suggest that saphenous vein phlebotomy causes less stress even when acclimation is not performed. Blood sampling in rodents is common practice in scientific studies. Some of the refined methods widely used are the puncture of the saphenous vein or tail vein, or even tail docking. The handling needs of these different blood sampling methods are different and can directly affect stress, increasing the variability of the study. Moreover, there is less aversion and stress if the animal is accustomed to the environment, handling and technique. Therefore, our study aimed to assess the influence of these three blood sampling techniques (saphenous puncture, tail vein puncture and tail vein docking) and the use of previous acclimation on different indicators of animal stress, assessing blood glucose concentrations and faecal corticosterone metabolites (FCMs). Twenty-four young adult male and female C57Bl6/J mice were divided in three groups by sampling method: tail docking (TD), saphenous vein puncture (SV) and caudal vein puncture (CV) groups. All mice were studied with and without acclimation, which was performed during 9 consecutive days. The results showed that both males and females present very similar responses to the different handling and sampling methods without significant differences. Nevertheless, acclimation in all sampling methods decreased glucose and FCM levels significantly. The method that obtained the lowest glucose and FCM levels with significance was saphenous vein puncture. Therefore, we can say that it causes less stress when performing prior acclimation, even when this involves greater handling of the animal. Our results contribute to refinement within the 3R concept and could serve researchers to programme and select a good handling technique and a welfare-friendly blood sampling method for their experiments. [ABSTRACT FROM AUTHOR]

The use of novel tools and technologies is transforming our approaches to study wild animal behaviour. Yet, from disturbance of natural habitats through field experiments to tagging, there is much ground for researchers to provide clear ethical assessment of their research practices and how they can affect wild animals.In this perspective, I review the current trends in field animal behaviour research, with a focus on studies on sociality, and investigate the forms under which such practices can impact animals. I focus particularly on the notions of disturbance and invasiveness.I build a parallel with the legal classifications in terms of degrees of severity found in the Swiss law and aimed at protecting animal well‐being, inspired by the principles of the 3R (Replace, Refine, Reduce) used in invasive animal research.I highlight how the impact of research can be assessed and monitored, particularly through behavioural and physiological means, identifying areas where such monitoring is critical, but also acknowledging the theoretical and practical limitations to such tools.I hope to trigger discussions on an ethical use of new tools and technologies, which can both inspire new research while limiting the disturbance to wild animals. [ABSTRACT FROM AUTHOR]

Government authorities often view the 3Rs of "replace, reduce, refine" popularized by Russell and Burch as both a regulatory principle and a governance principle aimed at reducing the total amount of animal distress in science. They thus expect that the 3Rs should, in time, result in changes in total animal experimentation numbers. Communications by Swiss authorities provide stark examples of this expectation. But the 3Rs do not aim at affecting animal experimentation at the level of total numbers; rather, they focus on study design in the individual case. While the underlying philosophy of the 3Rs indeed included a principle of seeking feasible overall reforms, this notion is completely absent in the 3Rs framework itself. Authorities need to stop treating the 3Rs as a means to reduce total distress and should instead invest resources into developing feasible and effective strategies for transformative governance in animal research. [ABSTRACT FROM AUTHOR]

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The circular economy (CE) can bring benefits but also pitfalls to the production processes, affecting a firm's economic performance. Using data from European SMEs, we empirically investigate, from the perspective of self‐determination theory, the extent to which the breadth of CE activities, that is, the number of CE activities undertaken by a firm, affects a firm's economic performance. Our study theorizes and shows that there is an inverted U‐shaped effect brought about by the number of CE activities on economic performance. This research advances our scientific understanding of the CE and provides managers with suggestions on how to maximize the benefits generated by the CE in terms of economic performance by implementing the right amount of CE activities. [ABSTRACT FROM AUTHOR]

Because low back pain is frequently a result of intervertebral disc degeneration (IVDD), strategies to regenerate or repair the IVD are currently being investigated. Often, ex vivo disc cultures of non-human IVD organs or tissue explants are used that usually do not exhibit natural IVDD. Therefore, degenerative changes mimicking those reported in human IVDD need to be induced. To support researchers in selecting ex vivo disc cultures, a systematic search was performed for them and their potential use for studying human IVDD reviewed. Five degeneration induction categories (proinflammatory cytokines, injury/damage, degenerative loading, enzyme, and other) were identified in 129 studies across 7 species. Methods to induce degeneration are diverse and can induce mild to severe degenerative changes that progress over time, as described for human IVDD. The induced degenerative changes are model-specific and there is no “one-fits-all” IVDD induction method. Nevertheless, specific aspects of human IVDD can be well mimicked. Currently, spontaneously degenerated disc cultures from large animals capture human IVDD in most aspects. Combinatorial approaches of several induction methods using discs derived from large animals are promising to recapitulate pathological changes on several levels, such as cellular behaviour, extracellular matrix composition, and biomechanical function, and therefore better mimic human IVDD. Future disc culture setups might increase in complexity, and mimic human IVDD even better. As ex vivo disc cultures have the potential to reduce and even replace animal trials, especially during preclinical development, advancement of such models is highly relevant for more efficient and cost-effective clinical translation from bench-to-bedside.

The use of nonhuman animals in biomedical research is regulated under stringent conditions, not only in response to societal attitudes towards animal experimentation but also because ethical responsibility in scientific research requires researchers and veterinarians to be more invested and aim to improve the welfare of animals used for experiments. Analyses of animal research oversight reveal the frequent approval of experiments, and the approval of some experiments has raised and continues to raise public concerns. Societal compliance is required for a consensus-based approach to animal research policy, prompting the need to have transparent discussions about oversight and the frequency of approvals. We discuss how frequent approval may be perceived and why it seems problematic from a societal perspective: the regulatory process exists to approve only legitimate experiments. Although some experiments remain unacceptable irrespective of their harm–benefit ratios, almost all experiments are approved. We explain some possible legitimate reasons for frequent approval and how the review process could be leading to the approval of illegitimate studies. To ensure transparency and improve public trust and understanding of oversight, we propose the adoption of a platform to inform society about how unethical experiments are screened out.

Simple Summary: Monitoring vitals sign such as the respiratory rate, heart rate, or temperature is of high importance to medical and biological research. Using camera-based methods, we monitored the respiratory rate of unconstrained laboratory rats by analyzing the visible breathing movement in the thorax. We hope this is a further step to enabling the non-invasive monitoring of rodent in an experimental environment without using implanted sensors, reducing the stress and pain within an otherwise unneeded operation. Animal research has always been crucial for various medical and scientific breakthroughs, providing information on disease mechanisms, genetic predisposition to diseases, and pharmacological treatment. However, the use of animals in medical research is a source of great controversy and ongoing debate in modern science. To ensure a high level of bioethics, new guidelines have been adopted by the EU, implementing the 3R principles to replace animal testing wherever possible, reduce the number of animals per experiment, and refine procedures to minimize stress and pain. Supporting these guidelines, this article proposes an improved approach for unobtrusive, continuous, and automated monitoring of the respiratory rate of laboratory rats. It uses the cyclical expansion and contraction of the rats' thorax/abdominal region to determine this physiological parameter. In contrast to previous work, the focus is on unconstrained animals, which requires the algorithms to be especially robust to motion artifacts. To test the feasibility of the proposed approach, video material of multiple rats was recorded and evaluated. High agreement was obtained between RGB imaging and the reference method (respiratory rate derived from electrocardiography), which was reflected in a relative error of 5.46%. The current work shows that camera-based technologies are promising and relevant alternatives for monitoring the respiratory rate of unconstrained rats, contributing to the development of new alternatives for a continuous and objective assessment of animal welfare, and hereby guiding the way to modern and bioethical research. [ABSTRACT FROM AUTHOR]

Introduction: Virtual Control Groups (VCGs) represent the concept of using historical control data from legacy animal studies to replace concurrent control group (CCG) animals. Based on the data curation and sharing activities of the Innovative Medicine Initiatives project eTRANSAFE (enhancing TRANSlational SAFEty Assessment through Integrative Knowledge Management) the ViCoG working group was established with the objectives of i) collecting suitable historical control data sets from preclinical toxicity studies, ii) evaluating statistical methodologies for building adequate and regulatory acceptable VCGs from historical control data, and iii) sharing those control-group data across multiple pharmaceutical companies. During the qualification process of VCGs a particular focus was put on the identification of hidden confounders in the data sets, which might impair the adequate matching of VCGs with the CCG. Methods: During our analyses we identified such a hidden confounder, namely, the choice of the anesthetic procedure used in animal experiments before blood withdrawal. Anesthesia using CO2 may elevate the levels of some electrolytes such as calcium in blood, while the use of isoflurane is known to lower these values. Identification of such hidden confounders is particularly important if the underlying experimental information (e.g., on the anesthetic procedure) is not routinely recorded in the standard raw data files, such as SEND (Standard for Exchange of Non-clinical Data). We therefore analyzed how the replacement of CCGs with VCGs would affect the reproducibility of treatment-related findings regarding electrolyte values (potassium, calcium, sodium, and phosphate). The analyses were performed using a legacy rat systemic toxicity study consisting of a control and three treatment groups conducted according to pertinent OECD guidelines. In the report of this study treatment-related hypercalcemia was reported. The rats in this study were anesthetized with isoflurane. Results: Replacing the CCGs with VCGs derived from studies comprising both anesthetics resulted in a shift of control electrolyte parameters. Instead of the originally reported hypercalcemia the use of VCG led to fallacious conclusions of no observed effect or hypocalcemia. Discussion: Our study highlights the importance of a rigorous statistical analysis including the detection and elimination of hidden confounders prior to the implementation of the VCG concept. [ABSTRACT FROM AUTHOR]

With the general goal to provide a rationale for the application of the 3Rs rule (Replace, Reduce, Refine), we aim to study the effect of the tumor microenvironment (TME) on the tumor response to a chemotherapeutic agent and use this research as a series of case studies in which to assess the application of the 3Rs rule from an epistemological point of view. In particular, by comparing reiterated experiments using 2D and 3D tumor cell cultures with murine models of cancer, we aim to assess to which extent mice can be Replaced or their number Reduced. In addition, we propose to further characterize a novel, Refined model of cancer that better mimics humans in respect to classical murine models. [ABSTRACT FROM AUTHOR]

Simple Summary: The chorioallantoic membrane (CAM) is a highly vascularized membrane found in avian eggs. Tumor cell lines can be grown on the CAM, which allows for the further analyses of the tumor grafts afterwards. We investigated the biological and growth characteristics of two breast cancer cell lines that resemble two biologically different breast cancer subgroups. Known biological features of the more aggressive breast cancer cell line were clearly confirmed in vitro and in the CAM model. Furthermore, the tissue-based pathological variables assessed in the CAM model were similar to those of the mouse xenografts and human patient tumor tissue. We suggest this in vivo model to be a reliable alternative for breast cancer research to reduce murine animal experiments. The chorioallantoic membrane (CAM) assay is an alternative in vivo model that allows for minimally invasive research of cancer biology. Using the CAM assay, we investigated phenotypical and functional characteristics (tumor grade, mitosis rate, tumor budding, hormone receptor (HR) and HER2 status, Ki-67 proliferation index) of two breast cancer cell lines, MCF-7 and MDA-MB-231, which resemble the HR+ (luminal) and triple-negative breast cancer (TNBC) subgroups, respectively. Moreover, the CAM results were directly compared with murine MCF-7- and MDA-MB-231-derived xenografts and human patient TNBC tissue. Known phenotypical and biological features of the aggressive triple-negative breast cancer cell line (MDA-MB-231) were confirmed in the CAM assay, and mouse xenografts. Furthermore, the histomorphological and immunohistochemical variables assessed in the CAM model were similar to those in human patient tumor tissue. Given the confirmation of the classical biological and growth properties of breast cancer cell lines in the CAM model, we suggest this in vivo model to be a reliable alternative test system for breast cancer research to reduce murine animal experiments. [ABSTRACT FROM AUTHOR]

There is a need for standardized in vitro models emulating the functionalities of the human intestinal tract to study human intestinal health without the use of laboratory animals. The Caco-2 cell line is a well-accepted and highly characterized intestinal barrier model, which has been intensively used to study intestinal (drug) transport, host–microbe interactions and chemical or drug toxicity. This cell line has been cultured in different in vitro models, ranging from simple static to complex dynamic microfluidic models. We aimed to investigate the effect of these different in vitro experimental variables on gene expression. To this end, we systematically collected and extracted data from studies in which transcriptome analyses were performed on Caco-2 cells grown on permeable membranes. A collection of 13 studies comprising 100 samples revealed a weak association of experimental variables with overall as well as individual gene expression. This can be explained by the large heterogeneity in cell culture practice, or the lack of adequate reporting thereof, as suggested by our systematic analysis of experimental parameters not included in the main analysis. Given the rapidly increasing use of in vitro cell culture models, including more advanced (micro) fluidic models, our analysis reinforces the need for improved, standardized reporting protocols. Additionally, our systematic analysis serves as a template for future comparative studies on in vitro transcriptome and other experimental data. [ABSTRACT FROM AUTHOR]

Permeability across cellular membranes is a key factor that influences absorption and distribution. Before absorption, many drugs must pass through the mucus barrier that covers all the wet surfaces of the human body. Cell-free in vitro tools currently used to evaluate permeability fail to effectively model the complexity of mucosal barriers. Here, we present an in vitro mucosal platform as a possible strategy for assessing permeability in a high-throughput setup. The PermeaPad 96-well plate was used as a permeability system and further coupled to a pathological, tridimensional mucus model. The physicochemical determinants predicting passive diffusion were determined by combining experimental and computational approaches. Drug solubility, size, and shape were found to be the critical properties governing permeability, while the charge of the drug was found to be influential on the interaction with mucus. Overall, the proposed mucosal platform could be a promising in vitro tool to model the complexity of mucosal tissues and could therefore be adopted for drug-permeability profiling. [ABSTRACT FROM AUTHOR]

Introduction: Chronic epilepsy models require neurosurgical procedures including depth electrode implants. The intrahippocampal kainate model is a frequently used chronic paradigm, which is based on chemoconvulsant administration and status epilepticus induction during the surgical procedure. This experimental approach raises the question of the extent to which this approach affects postsurgical recovery. In addition to the short- and long-term impact of the surgical intervention, a potential impact of highly frequent electrographic seizure events needs to be considered in the context of severity assessment. Methods: Various behavioral, biochemical, and telemetric parameters were analyzed in four experimental groups of mice: 1st naive, 2nd with transmitter implants, 3rd with transmitter and electrode implants, and 4th with transmitter implants, electrode implants, and kainate-induced status epilepticus. Results: During the early postsurgical phase, transmitter implants caused a transient impact on Mouse Grimace scores and intragroup increase of fecal corticosterone metabolites. Additional craniotomy was associated with an influence on total heart rate variability and fecal corticosterone metabolites. Heart rate and Irwin score increases as well as a prolonged increase in Mouse Grimace scores pointed to an added burden related to the induction of a nonconvulsive status epilepticus. Data from the chronic phase argued against a relevant influence of frequent electrographic seizures on behavioral patterns, fecal corticosterone metabolites, heart rate, and its variability. However, Irwin scores indicated long-term changes in some animals with increased reactivity, body tone, and Straub tail. Interestingly, selected behavioral and telemetric data from the early post-status epilepticus phase correlated with the frequency of electrographic seizure events in the chronic phase. Conclusion: In conclusion, our findings argue against the pronounced impact of highly frequent electrographic seizures on the well-being of mice. However, an increased level of nervousness in a subgroup of animals should be considered for handling procedures and refinement measures. In the early postsurgical phase, several parameters indicate an influence of the interventions with evidence that the nonconvulsive status epilepticus can negatively affect the recovery. Thus, the development and validation of refinement efforts should focus on this experimental phase. Finally, the datasets suggest that simple readout parameters may predict the long-term consequences of the epileptogenic insult. Respective biomarker candidates require further validation in the follow-up studies in models with subgroups of animals with or without epilepsy development. [ABSTRACT FROM AUTHOR]

Preclinical cardiovascular research relies heavily on non-invasive in-vivo echocardiography in mice and rats to assess cardiac function and morphology, since the complex interaction of heart, circulation, and peripheral organs are challenging to mimic ex-vivo. While n-numbers of annually used laboratory animals worldwide approach 200 million, increasing efforts are made by basic scientists aiming to reduce animal numbers in cardiovascular research according to the 3R’s principle. The chicken egg is well-established as a physiological correlate and model for angiogenesis research but has barely been used to assess cardiac (patho-) physiology. Here, we tested whether the established in-ovo system of incubated chicken eggs interfaced with commercially available small animal echocardiography would be a suitable alternative test system in experimental cardiology. To this end, we defined a workflow to assess cardiac function in 8–13-day-old chicken embryos using a commercially available high resolution ultrasound system for small animals (Vevo 3100, Fujifilm Visualsonics Inc.) equipped with a high frequency probe (MX700; centre transmit: 50 MHz). We provide detailed standard operating procedures for sample preparation, image acquisition, data analysis, reference values for left and right ventricular function and dimensions, and inter-observer variabilities. Finally, we challenged incubated chicken eggs with two interventions well-known to affect cardiac physiology—metoprolol treatment and hypoxic exposure—to demonstrate the sensitivity of in-ovo echocardiography. In conclusion, in-ovo echocardiography is a feasible alternative tool for basic cardiovascular research, which can easily be implemented into the small animal research environment using existing infrastructure to replace mice and rat experiments, and thus, reduce use of laboratory animals according to the 3R principle. [ABSTRACT FROM AUTHOR]