| 1. |
- Adamson, L, et al.
(author)
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Insulin and IGF-1 mediated inhibition of apoptosis in CHO cells grown in suspension in a protein-free medium
- 2007
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In: Alternatives to laboratory animals : ATLA. - : SAGE Publications. - 0261-1929 .- 2632-3559. ; 35:3, s. 349-352
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Journal article (peer-reviewed)abstract
- When Chinese hamster ovary (CHO) cells were grown in suspension and deprived of serum, 40% of them became apoptotic after 72 hours, as determined by flow cytometry analysis of TUNEL-labelled cells. Cell viability, assessed by erythrocin B staining, decreased correspondingly. An increase in the total fraction of cells expressing interleukin converting enzyme (ICE; caspase 1), B-cell lymphoma 2 protein (Bcl-2,) and Bcl-2 associated x protein (Bax) was shown by antibody probing and subsequent flow cytometry. The p53 tumour suppressor gene product level remained low within the cell population. Insulin-like growth factor-1 (IGF-1) inhibited cell death in a concentration-dependent manner, and at 20ng/ml, cell viability was maintained close to 100% and no apoptotic cells were detected. Also, insulin was shown to inhibit cell death — at 1.0μg/ml, cell viability was 95%, whereas 10% of the cells stained for apoptosis. At the highest concentrations of IGF-1 and insulin, the expression of ICE, Bcl-2 and Bax was fully suppressed, whereas the p53 product level increased, despite still being detectable in a minority of cells. Under these conditions, IGF-1 may increase p53 expression to restrain abnormal cell proliferation. It is concluded that special attention should be paid to exposure and culture conditions that induce acquired susceptibility to a toxic insult, during the development and validation of cell-based assays.
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| 2. |
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| 3. |
- Andersson, Martin, et al.
(author)
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In Silico Prediction of Eye Irritation Using Hansen Solubility Parameters and Predicted pKa Values
- 2023
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In: ATLA (Alternatives to Laboratory Animals). - : SAGE Publications Inc.. - 0261-1929. ; 51:3, s. 204-
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Journal article (peer-reviewed)abstract
- An in silico method has been developed that permits the binary differentiation between pure liquids causing serious eye damage or eye irritation, and pure liquids with no need for such classification, according to the UN GHS system. The method is based on the finding that the Hansen Solubility Parameters (HSP) of a liquid are collectively important predictors for eye irritation. Thus, by applying a two-tier approach in which in silico predicted pKa values (firstly) and a trained model based solely on in silico-predicted HSP data (secondly) were used, we have developed, and validated, a fully in silico approach for predicting the outcome of a Draize test (in terms of UN GHS Cat. 1/Cat. 2A/Cat. 2B or UN GHS No Cat.) with high validation set performance (sensitivity = 0.846, specificity = 0.818, balanced accuracy = 0.832) using SMILES only. The method is applicable to pure non-ionic liquids with molecular weight below 500 g/mol, fewer than six hydrogen bond donors (e.g. nitrogen–hydrogen or oxygen–hydrogen bonds) and fewer than eleven hydrogen bond acceptors (e.g. nitrogen or oxygen atoms). Due to its fully in silico characteristics, this method can be applied to pure liquids that are still at the desktop design stage and not yet in production.
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| 4. |
- Baumans, V
(author)
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Methods for evaluation of laboratory animal well-being
- 2004
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In: Alternatives to laboratory animals : ATLA. - : SAGE Publications. - 0261-1929 .- 2632-3559. ; 3232 Suppl 1A, s. 161-162
-
Journal article (peer-reviewed)abstract
- Well-being is a relative concept, referring to the state of an animal in relation to its ability to cope with its environment. This ability to cope is what we usually try to measure when evaluating the animal's well-being. Good welfare is, in general, considered to be related to a broad behavioural repertoire, which requires a considerable knowledge of the animal's species-specific behaviour and their basic biology. Ideally, well-being should be measured in a positive way, such as measuring pleasure by anticipatory behaviour. However, parameters have more often been designed for detecting failures to cope, leading to stress and/or discomfort. Parameters used in the assessment of discomfort are behavioural parameters, such as stereotypies, reduction in grooming, changes in activity; physiological parameters, such as body weight, abnormal posture, respiratory signs, heart rate, hormone levels; and post-mortem signs, as retrospective parameters, such as stomach ulcers, adrenal cortex size, fatty deposits. The usefulness of these parameters is discussed.
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| 5. |
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| 6. |
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| 7. |
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| 8. |
- Brandmaier, Stefan, et al.
(author)
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The QSPR-THESAURUS : The Online Platform of the CADASTER Project
- 2014
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In: ATLA (Alternatives to Laboratory Animals). - : SAGE Publications. - 0261-1929. ; 42:1, s. 13-24
-
Journal article (peer-reviewed)abstract
- The aim of the CADASTER project (CAse Studies on the Development and Application of in Silico Techniques for Environmental Hazard and Risk Assessment) was to exemplify REACH-related hazard assessments for four classes of chemical compound, namely, polybrominated diphenylethers, per and polyfluorinated compounds, (benzo)triazoles, and musks and fragrances. The QSPR-THESAURUS website (http: / /qspr-thesaurus.eu) was established as the project's online platform to upload, store, apply, and also create, models within the project. We overview the main features of the website, such as model upload, experimental design and hazard assessment to support risk assessment, and integration with other web tools, all of which are essential parts of the QSPR-THESAURUS.
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| 9. |
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| 10. |
- Burt, Tal, et al.
(author)
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Phase 0, Including Microdosing Approaches : Applying the Three Rs and Increasing the Efficiency of Human Drug Development
- 2018
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In: ATLA (Alternatives to Laboratory Animals). - : FRAME. - 0261-1929. ; 46:6, s. 335-346
-
Journal article (peer-reviewed)abstract
- Phase 0 approaches, including microdosing, involve the use of sub-therapeutic exposures to the tested drugs, thus enabling safer, more-relevant, quicker and cheaper first-in-human (FIH) testing. These approaches also have considerable potential to limit the use of animals in human drug development. Recent years have witnessed progress in applications, methodology, operations, and drug development culture. Advances in applications saw an expansion in therapeutic areas, developmental scenarios and scientific objectives, in, for example, protein drug development and paediatric drug development. In the operational area, the increased sensitivity of Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS), expansion of the utility of Positron Emission Tomography (PET) imaging, and the introduction of Cavity Ring-Down Spectroscopy (CRDS), have led to the increased accessibility and utility of Phase 0 approaches, while reducing costs and exposure to radioactivity. PET has extended the application of microdosing, from its use as a predominant tool to record pharmacokinetics, to a method for recording target expression and target engagement, as well as cellular and tissue responses. Advances in methodology include adaptive Phase 0/Phase 1 designs, cassette and cocktail microdosing, and Intra-Target Microdosing (ITM), as well as novel modelling opportunities and simulations. Importantly, these methodologies increase the predictive power of extrapolation from microdose to therapeutic level exposures. However, possibly the most challenging domain in which progress has been made, is the culture of drug development. One of the main potential values of Phase 0 approaches is the opportunity to terminate development early, thus not only applying the principle of 'kill-early-kill-cheap' to enhance the efficiency of drug development, but also obviating the need for the full package of animal testing required for therapeutic level Phase 1 studies. Finally, we list developmental scenarios that utilised Phase 0 approaches in novel drug development.
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| 11. |
- Carbone, L, et al.
(author)
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Report of the workshop on euthanasia guidelines and practices
- 2004
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In: Alternatives to laboratory animals : ATLA. - : SAGE Publications. - 0261-1929 .- 2632-3559. ; 3232 Suppl 1B, s. 445-446
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Journal article (other academic/artistic)abstract
- Determining ethical standards for laboratory animal euthanasia requires an assessment of the relative amounts of pain and distress caused by different methods. Animal behaviour data are an important indicator of pain and distress, but their interpretation can be controversial; moreover, behaviour is more easily assessed with some euthanasia methods than with others. While every euthanasia method requires careful study, CO2 inhalation has come under close scrutiny both because it is so widely used for rodent euthanasia, and because it has, until recently, long been considered relatively non-aversive.
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| 12. |
- Cassani, Stefano, et al.
(author)
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Evaluation of CADASTER QSAR Models for the Aquatic Toxicity of (Benzo)triazoles and Prioritisation by Consensus Prediction
- 2013
-
In: ATLA (Alternatives to Laboratory Animals). - : SAGE Publications. - 0261-1929. ; 41:1, s. 49-64
-
Journal article (peer-reviewed)abstract
- QSAR regression models of the toxicity of triazoles and benzotriazoles ([B] TAZs) to an alga (Pseudokirchneriella subcapitata), Daphnia magna and a fish (Onchorhynchus mykiss), were developed by five partners in the FP7-EU Project, CADASTER. The models were developed by different methods - Ordinary Least Squares (OLS), Partial Least Squares (PLS), Bayesian regularised regression and Associative Neural Network (ASNN) - by using various molecular descriptors (DRAGON, PaDEL-Descriptor and QSPR-THESAURUS web). In addition, different procedures were used for variable selection, validation and applicability domain inspection. The predictions of the models developed, as well as those obtained in a consensus approach by averaging the data predicted from each model, were compared with the results of experimental tests that were performed by two CADASTER partners. The individual and consensus models were able to correctly predict the toxicity classes of the chemicals tested in the CADASTER project, confirming the utility of the QSAR approach. The models were also used for the prediction of aquatic toxicity of over 300 (B)TAZs, many of which are included in the REACH pre-registration list, and were without experimental data. This highlights the importance of QSAR models for the screening and prioritisation of untested chemicals, in order to reduce and focus experimental testing.
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| 13. |
- Ceder, R, et al.
(author)
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The application of normal, SV40 T-antigen-immortalised and tumour-derived oral keratinocytes, under serum-free conditions, to the study of the probability of cancer progression as a result of environmental exposure to chemicals
- 2007
-
In: Alternatives to laboratory animals : ATLA. - : SAGE Publications. - 0261-1929 .- 2632-3559. ; 35:6, s. 621-639
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Journal article (peer-reviewed)abstract
- In vitro models are currently not considered to be suitable replacements for animals in experiments to assess the multiple factors that underlie the development of cancer as a result of environmental exposure to chemicals. An evaluation was conducted on the potential use of normal keratinocytes, the SV40 T-antigen-immortalised keratinocyte cell line, SVpgC2a, and the carcinoma cell line, SqCC/Y1, alone and in combination, and under standardised serum-free culture conditions, to study oral cancer progression. In addition, features considered to be central to cancer development as a result of environmental exposure to chemicals, were analysed. Genomic expression, and enzymatic and functional data from the cell lines reflected many aspects of the transition of normal tissue epithelium, via dysplasia, to full malignancy. The composite cell line model develops aberrances in proliferation, terminal differentiation and apoptosis, in a similar manner to oral cancer progression in vivo. Transcript and protein profiling links aberrations in multiple gene ontologies, molecular networks and tumour biomarker genes (some proposed previously, and some new) in oral carcinoma development. Typical specific changes include the loss of tumour-suppressor p53 function and of sensitivity to retinoids. Environmental agents associated with the aetiology of oral cancer differ in their requirements for metabolic activation, and cause toxic effects to cells in both the normal and the transformed states. The results suggest that the model might be useful for studies on the sensitivity of cells to chemicals at different stages of cancer progression, including many aspects of the integrated roles of cytotoxicity and genotoxicity. Overall, the properties of the SVpgC2a and SqCC/Y1 cell lines, relative to normal epithelial cells in monolayer or organotypic culture, support their potential applicability to mechanistic studies on cancer risk factors, including, in particular, the definition of critical toxicity effects and dose–effect relationships.
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| 14. |
- Clemedson, Cecilia, et al.
(author)
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Development of an in vitro test battery for the estimation of acute human systemic toxicity : An outline of the EDIT project. Evaluation-guided Development of New In Vitro Test Batteries
- 2002
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In: ATLA (Alternatives to Laboratory Animals). - 0261-1929. ; 30:3, s. 313-321
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Journal article (peer-reviewed)abstract
- The aim of the Evaluation-guided Development of new In Vitro Test Batteries (EDIT) multicentre programme is to establish and validate in vitro tests relevant to toxicokinetics and for organ-specific toxicity, to be incorporated into optimal test batteries for the estimation of human acute systemic toxicity. The scientific basis of EDIT is the good prediction of human acute toxicity obtained with three human cell line tests (R(2) = 0.77), in the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme. However, the results from the MEIC study indicated that at least two other types of in vitro test ought to be added to the existing test battery to improve the prediction of human acute systemic toxicity - to determine key kinetic events (such as biotransformation and passage through biological barriers), and to predict crucial organ-specific mechanisms not covered by the tests in the MEIC battery. The EDIT programme will be a case-by-case project, but the establishment and validation of new tests will be carried through by a common, step-wise procedure. The Scientific Committee of the EDIT programme defines the need for a specific set of toxicity or toxicokinetic data. Laboratories are then invited to perform the defined tests in order to provide the "missing" data for the EDIT reference chemicals. The results obtained will be evaluated against the MEMO (the MEIC Monograph programme) database, i.e. against human acute systemic lethal and toxicity data. The aim of the round-table discussions at the 19th Scandinavian Society for Cell Toxicology (SSCT) workshop, held in Ringsted, Denmark on 6-9 September 2001, was to identify which tests are the most important for inclusion in the MEIC battery, i.e. which types of tests the EDIT programme should focus on. It was proposed that it is important to include in vitro methods for various kinetic events, such as biotransformation, absorption in the gut, passage across the blood-brain barrier, distribution volumes, protein binding, and renal clearance/accumulation. Models for target organ toxicity were also discussed. Because several of the outlier chemicals (paracetamol, digoxin, malathion, nicotine, paraquat, atropine and potassium cyanide) in the MEIC in vivo-in vitro evaluation have a neurotoxic potential, it was proposed that the development within the EDIT target organ programme should initially be focused on the nervous system.
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| 15. |
- Clemedson, Cecilia, et al.
(author)
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The integrated acute systemic toxicity project (ACuteTox) for the optimisation and validation of alternative in vitro tests.
- 2007
-
In: ATLA (Alternatives to Laboratory Animals). - 0261-1929. ; 35:1, s. 33-8
-
Journal article (peer-reviewed)abstract
- The ACuteTox project is designed to replace animal testing for acute systemic toxicity, as is widely used today for regulatory purposes, by using in vitro and in silico alternatives. In spite of the fact that earlier studies on acute systemic toxicity demonstrated a good correlation between in vitro basal cytotoxicity data (the 50% inhibitory concentration [IC50]) in human cell lines and rodent LD50 values, and an even better correlation between IC50 values and human lethal blood concentrations, very few non-animal tests have been accepted for general use. Therefore, the aim of the ACuteTox project is to adapt new testing strategies, for example, the implementation of new endpoints and new cell systems for toxicity screening, organ-specific models, metabolism-dependent toxicity, tissue absorption, distribution and excretion, and computer-based prediction models. A new database, AcuBase, containing descriptions and results of in vitro tests of the 97 reference chemicals, as well as the results of animal experimentation, and human acute toxicity data, will be generated within the framework of ACuteTox. Scientists from 13 European countries are working together and making efforts to find the most appropriate testing strategies for the prediction of human acute systemic toxicity, and also to select a robust in vitro test battery for cytotoxicity testing of chemicals.
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| 16. |
- Clothier, Richard, et al.
(author)
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A database of IC50 values and principal component analysis of results from six basal cytotoxicity assays, for use in the modelling of the in vivo and in vitro data of the EU ACuteTox project.
- 2008
-
In: Altern Lab Anim. - 0261-1929. ; 36:5, s. 503-19
-
Journal article (peer-reviewed)abstract
- The main aim of the ACuteTox project (part of the EU 6th Framework programme) is to demonstrate that animal tests for acute systemic toxicity can be replaced by alternative in vitro assays. In this project, data for 97 reference chemicals were collected in the AcuBase database, designed to handle deposited in vitro and in vivo (human and animal) data. To demonstrate the applicability of in vitro basal cytotoxicity tests and in vitro-in vivo modelling, it was deemed necessary to obtain data that were generated via defined standard operating procedures. The molar basal cytotoxicity IC50 values (the 50% inhibitory concentrations for the endpoint measured) for a mouse fibroblast cell line (3T3), a human hepatic cell line (HepG2), a rat hepatic cell line (Fa32), and a human neutrophil cell line (HL-60), were compared, and gave an R(2) correlation of 0.83. To identify chemicals that showed differential cytotoxicity to the various cell types involved, principal component analysis (PCA) was undertaken independently, once all the results had been returned. This showed that colchicine, cycloheximide, digoxin, 5-fluorouracil and hexachlorobenzene gave the lowest correlations with the first score vector of the PCA. The results presented are to be used to identify outliers that need to be further studied via the use of tissue-specific in vitro assays.
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| 17. |
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| 18. |
- Durjava, Mojca Kos, et al.
(author)
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Experimental Assessment of the Environmental Fate and Effects of Triazoles and Benzotriazole
- 2013
-
In: ATLA (Alternatives to Laboratory Animals). - : SAGE Publications. - 0261-1929. ; 41:1, s. 65-75
-
Journal article (peer-reviewed)abstract
- The environmental fate and effects of triazoles and benzotriazoles are of concern within the context of chemical regulation. As part of an intelligent testing strategy, experimental tests were performed on endpoints that are relevant for risk assessment. The experimental tests included the assessment of eco-toxicity to an alga, a daphnid and zebrafish embryos, and the assessment of ready biodegradability. Triazole and benzotriazole compounds were selected for testing, based on existing toxicity data for vertebrate and invertebrate species, as well as on the principal component analysis of molecular descriptors aimed at selecting the minimum number of test compounds in order to maximise the chemical domain spanned for both compound classes. The experimental results show that variation in the toxicities of triazoles and benzotriazole across species was relatively minor; in general, the largest factor was approximately 20. The study conducted indicated that triazoles are not readily biodegradable.
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| 19. |
- Ekwall, Björn, et al.
(author)
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MEIC evaluation of acute systemic toxicity : Part VIII. Multivariate partial least squares evaluation, including the selection of a battery of cell line tests with a good prediction of human acute lethal peak blood concentrations for 50 chemicals
- 2000
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In: Atla-Alternatives to Laboratory Animals. - 0261-1929. ; 28, s. 201-34
-
Journal article (peer-reviewed)abstract
- The Multicenter Evaluation of In vitro Cytotoxicity (MEIC) programme was set up to evaluate the relevance for human acute toxicity of in vitro cytotoxicity tests. A total of 61 assays were used to test all 50 reference chemicals. The results of all the tests and the human database were presented in the first five papers of this series. An evaluation of the relevance for human acute toxicity of all submitted test results with use of hard linear regression modelling was presented in the next two papers, and demonstrated a high relevance of in vitro tests, notably tests involving human cell lines. In the present study, multivariate partial least square (PLS) modelling with latent variables analysis has been used to reach two objectives. The first objective was to study the prediction of human acute toxicity by the 61 assays. The second objective was to select a practical battery from the 61 assays, with an optimal prediction of lethal blood concentrations from human acute poisonings of the chemicals. A two-component PLS model of all 61 assays predicted three sets of lethal blood concentrations (clinical, forensic and peak concentrations) very well (R-2 = 0.77, 0.76 and 0.83, Q(2) = 0.74, 0.72 and 0.81, respectively), providing correlative evidence for a high relevance for human acute toxicity of most of the assays. The assays with human cells were highly predictive, whereas assays with Very short incubation times and non-fish ecotoxicological assays were least predictive. These findings confirm the previous results from linear regression analysis. To select an optimal battery, 24 successive PLS models of in vitro data were compared with lethal peak concentrations. The battery selection was based on 38 chemicals with reliable and relevant lethal peak concentrations. An initial PLS model of all 61 assays was used to select the 15 most predictive and most distinct assays. Subsequent PLS models were used to measure the decrease in prediction when assays were deleted from the 15-test battery, as well as the increase in prediction when some extra-predictive assays (as identified by the deletion process) were added later to an optimal two-test battery. The most predictive three-test battery (R-2 = 0.79 and Q(2) = 0.78 for all 50 chemicals) included two circumstantial assays. The most predictive and most cost-effective battery consisted of three human cell line assays, with four endpoints and two exposure times, i.e. protein content (24 hours), ATP content (24 hours), inhibition of elongation of cells (24 hours), and pH-change (7 days). This 1, 5, 9, 16 battery exclusively measures basal cytotoxicity, and is highly predictive (R-2 = 0.77 and Q(2) = 0.76 for 50 chemicals) of the actual lethal peak blood concentrations from acute poisonings in humans. The battery prediction compares favourably with the prediction of human lethal dose by a PLS model of rat and mouse 50% lethal dose (LD50) values for the 50 chemicals (R-2 = 0.65 and Q(2) = 0.64). The three assays of the battery and other promising MEIC assays should be formally validated as soon as possible. The battery can be used immediately for several non-regulatory purposes, including the high-throughput screening of potential pharmaceuticals.
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| 20. |
- Fagerholm, Urban, et al.
(author)
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In Silico Prediction of Human Clinical Pharmacokinetics with ANDROMEDA by Prosilico : Predictions for an Established Benchmarking Data Set, a Modern Small Drug Data Set, and a Comparison with Laboratory Methods
- 2023
-
In: ATLA (Alternatives to Laboratory Animals). - : SAGE Publications. - 0261-1929. ; 51:1, s. 39-54
-
Journal article (peer-reviewed)abstract
- There is an ongoing aim to replace animal and in vitro laboratory models with in silico methods. Such replacement requires the successful validation and comparably good performance of the alternative methods. We have developed an in silico prediction system for human clinical pharmacokinetics, based on machine learning, conformal prediction and a new physiologically-based pharmacokinetic model, i.e. ANDROMEDA. The objectives of this study were: a) to evaluate how well ANDROMEDA predicts the human clinical pharmacokinetics of a previously proposed benchmarking data set comprising 24 physicochemically diverse drugs and 28 small drug molecules new to the market in 2021; b) to compare its predictive performance with that of laboratory methods; and c) to investigate and describe the pharmacokinetic characteristics of the modern drugs. Median and maximum prediction errors for the selected major parameters were ca 1.2 to 2.5-fold and 16-fold for both data sets, respectively. Prediction accuracy was on par with, or better than, the best laboratory-based prediction methods (superior performance for a vast majority of the comparisons), and the prediction range was considerably broader. The modern drugs have higher average molecular weight than those in the benchmarking set from 15 years earlier (ca 200 g/mol higher), and were predicted to (generally) have relatively complex pharmacokinetics, including permeability and dissolution limitations and significant renal, biliary and/or gut-wall elimination. In conclusion, the results were overall better than those obtained with laboratory methods, and thus serve to further validate the ANDROMEDA in silico system for the prediction of human clinical pharmacokinetics of modern and physicochemically diverse drugs.
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| 21. |
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| 22. |
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| 23. |
- Grafström, Roland C, et al.
(author)
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Toward the Replacement of Animal Experiments through the Bioinformatics-driven Analysis of 'Omics' Data from Human Cell Cultures
- 2015
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In: ATLA (Alternatives to Laboratory Animals). - : SAGE Publications. - 0261-1929 .- 2632-3559. ; 43:5, s. 325-332
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Journal article (peer-reviewed)abstract
- This paper outlines the work for which Roland Grafström and Pekka Kohonen were awarded the 2014 Lush Science Prize. The research activities of the Grafström laboratory have, for many years, covered cancer biology studies, as well as the development and application of toxicity-predictive in vitro models to determine chemical safety. Through the integration of in silico analyses of diverse types of genomics data (transcriptomic and proteomic), their efforts have proved to fit well into the recently-developed Adverse Outcome Pathway paradigm. Genomics analysis within state-of-the-art cancer biology research and Toxicology in the 21st Century concepts share many technological tools. A key category within the Three Rs paradigm is the Replacement of animals in toxicity testing with alternative methods, such as bioinformatics-driven analyses of data obtained from human cell cultures exposed to diverse toxicants. This work was recently expanded within the pan-European SEURAT-1 project (Safety Evaluation Ultimately Replacing Animal Testing), to replace repeat-dose toxicity testing with data-rich analyses of sophisticated cell culture models. The aims and objectives of the SEURAT project have been to guide the application, analysis, interpretation and storage of 'omics' technology-derived data within the service-oriented sub-project, ToxBank. Particularly addressing the Lush Science Prize focus on the relevance of toxicity pathways, a 'data warehouse' that is under continuous expansion, coupled with the development of novel data storage and management methods for toxicology, serve to address data integration across multiple 'omics' technologies. The prize winners' guiding principles and concepts for modern knowledge management of toxicological data are summarised. The translation of basic discovery results ranged from chemical-testing and material-testing data, to information relevant to human health and environmental safety.
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| 24. |
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| 25. |
- Hedberg, JJ, et al.
(author)
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Uniform expression of alcohol dehydrogenase 3 in epithelia regenerated with cultured normal, immortalised and malignant human oral keratinocytes
- 2001
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In: Alternatives to laboratory animals : ATLA. - : SAGE Publications. - 0261-1929 .- 2632-3559. ; 29:3, s. 325-333
-
Journal article (peer-reviewed)abstract
- The human oral epithelium is a target for damage from the inhalation of formaldehyde. However, most experimental studies on this chemical have relied on laboratory animals that are obligatory nose breathers, including rats and mice. Therefore, in vitro model systems that mimic the structure of the human oral epithelium and which retain normal tissue-specific metabolic competence are desirable. Based on the established role of alcohol dehydrogenase 3 (ADH3), also known as glutathione-dependent formaldehyde dehydrogenase, as the primary enzyme catalysing the detoxification of formaldehyde, the aim of this study was to investigate the expression of ADH3 in organotypic epithelia regenerated with normal (NOK), immortalised (SVpgC2a) and malignant (SqCC/Y1) human oral keratinocytes. Organotypic epithelia, usually consisting of 5–10 cell layers, were produced at the air–liquid interface of collagen gels containing human oral fibroblasts, after culture for 10 days in a standardised serum-free medium. Immunochemical staining demonstrated uniform expression of ADH3 in these organotypic epithelia, as well as in the epithelial cells of oral tissue. The specificity of the ADH3 antiserum was ascertained from the complete neutralisation of the immunochemical reaction with purified ADH3 protein. Assessment of the staining intensities indicated that the expression levels were similar among the regenerated epithelia. Furthermore, the regenerated epithelia showed similar ADH3 expression to the epithelium in oral tissue. Therefore, a tissue-like expression pattern for ADH3 can be generated from the culture of various oral keratinocyte lines in an organotypic state. Similar expression levels among the various cell lines indicate the preservation of ADH3 during malignant transformation, and therefore that NOK, SVpgC2a and SqCC/Y1 represent functional models for in vitro studies of formaldehyde metabolism in human oral mucosa.
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| 26. |
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| 27. |
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| 28. |
- Kramer, K, et al.
(author)
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Effect of conditioning on the increase of heart rate and body temperature provoked by handling in the mouse
- 2004
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In: Alternatives to laboratory animals : ATLA. - : SAGE Publications. - 0261-1929 .- 2632-3559. ; 3232 Suppl 1A, s. 177-181
-
Journal article (peer-reviewed)abstract
- To assess the effect of procedures on animal welfare, various physiological parameters, such as body weight, hormone levels in plasma and/or urine, heart rate (HR), blood pressure and body temperature (BT), can be used. When measuring physiological parameters with techniques involving restraint of the animals, the results must be interpreted with caution, since restraint itself may have an effect on those parameters. Radio-telemetry, using an implantable transmitter, provides a way to obtain more accurate and reliable physiological measurements from freely moving animals in their own environment. In this study, we have used radio-telemetry to investigate the influence of conditioning on the increase of HR and BT as provoked by handling of mice. It was found that, after a conditioning period of 12 days, the increase of HR due to handling was significantly reduced.
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| 29. |
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| 30. |
- Mandenius, Carl-Fredrik, et al.
(author)
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Toward Preclinical Predictive Drug Testing for Metabolism and Hepatotoxicity by Using In Vitro Models Derived from Human Embryonic Stem Cells and Human Cell Lines - A Report on the Vitrocellomics EU-project
- 2011
-
In: ATLA-ALTERNATIVES TO LABORATORY ANIMALS. - : Fund for Replacement of Animals in Medical Experiments; 1999. - 0261-1929 .- 2632-3559. ; 39:2, s. 147-171
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Journal article (peer-reviewed)abstract
- Drug-induced liver injury is a common reason for drug attrition in late clinical phases, and even for post-launch withdrawals. As a consequence, there is a broad consensus in the pharmaceutical industry, and within regulatory authorities, that a significant improvement of the current in vitro test methodologies for accurate assessment and prediction of such adverse effects is needed. For this purpose, appropriate in vivo-like hepatic in vitro models are necessary, in addition to novel sources of human hepatocytes. In this report, we describe recent and ongoing research toward the use of human embryonic stem cell (hESC)-derived hepatic cells, in conjunction with new and improved test methods, for evaluating drug metabolism and hepatotoxicity. Recent progress on the directed differentiation of human embryonic stem cells to the functional hepatic phenotype is reported, as well as the development and adaptation of bioreactors and toxicity assay technologies for the testing of hepatic cells. The aim of achieving a testing platform for metabolism and hepatotoxicity assessment, based on hESC-derived hepatic cells, has advanced markedly in the last 2-3 years. However, great challenges still remain, before such new test systems could be routinely used by the industry. In particular, we give an overview of results from the Vitrocellomics project (EU Framework 6) and discuss these in relation to the current state-of-the-art and the remaining difficulties, with suggestions on how to proceed before such in vitro systems can be implemented in industrial discovery and development settings and in regulatory acceptance.
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| 31. |
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| 32. |
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| 33. |
- Sahlin, Ullrika, et al.
(author)
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Arguments for considering uncertainty in QSAR predictions in hazard and risk assessments
- 2013
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In: ATLA (Alternatives to Laboratory Animals). - : SAGE Publications. - 0261-1929. ; 41:1, s. 91-110
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Journal article (peer-reviewed)abstract
- Chemical regulation allows non-in vivo testing (i.e. in silico-derived and in vitro-derived) information to replace experimental values from in vivo studies in hazard and risk assessments. Although non-in vitro testing information on chemical activities or properties is subject to added uncertainty as compared to in vivo testing information, this uncertainty is commonly not (fully) taken into account. Considering uncertainty in predictions from quantitative structure-activity relationships (QSARs), which are a form of non-in vivo testing information, may improve the way that QSARs support chemical safety assessment under the EU Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) system. We argue that it is useful to consider uncertainty in QSAR predictions, as it: a) supports rational decision-making; b) facilitates cautious risk management; c) informs uncertainty analysis in probabilistic risk assessment; d) may aid the evaluation of QSAR predictions in weight-of-evidence approaches; and e) provides a probabilistic model to verify the experimental data used in risk assessment. The discussion is illustrated by using case studies of QSAR integrated hazard and risk assessment from the EU-financed CADASTER project.
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| 34. |
- Sahlin, Ullrika
(author)
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Uncertainty in QSAR Predictions
- 2013
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In: ATLA (Alternatives to Laboratory Animals). - : SAGE Publications. - 0261-1929. ; 41:1, s. 111-125
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Journal article (peer-reviewed)abstract
- It is relevant to consider uncertainty in individual predictions when quantitative structure-activity (or property) relationships (QSARs) are used to support decisions of high societal concern. Successful communication of uncertainty in the integration of QSARs in chemical safety assessment under the EU Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) system can be facilitated by a common understanding of how to define, characterise, assess and evaluate uncertainty in QSAR predictions. A QSAR prediction is, compared to experimental estimates, subject to added uncertainty that comes from the use of a model instead of empirically-based estimates. A framework is provided to aid the distinction between different types of uncertainty in a QSAR prediction: quantitative, i.e. for regressions related to the error in a prediction and characterised by a predictive distribution; and qualitative, by expressing our confidence in the model for predicting a particular compound based on a quantitative measure of predictive reliability. It is possible to assess a quantitative (i.e. probabilistic) predictive distribution, given the supervised learning algorithm, the underlying QSAR data, a probability model for uncertainty and a statistical principle for inference. The integration of QSARs into risk assessment may be facilitated by the inclusion of the assessment of predictive error and predictive reliability into the "unambiguous algorithm", as outlined in the second OECD principle.
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| 35. |
- Sarang, Z, et al.
(author)
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Microarray assessment of fibronectin, collagen and integrin expression and the role of fibronectin-collagen coating in the growth of normal, SV40 T-antigen-immortalised and malignant human oral keratinocytes
- 2003
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In: Alternatives to laboratory animals : ATLA. - : SAGE Publications. - 0261-1929 .- 2632-3559. ; 31:6, s. 575-585
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Journal article (peer-reviewed)abstract
- Extracellular matrix proteins affect the growth and survival of epithelial tissues. Accordingly, surface coating with fibronectin and collagen is a common practice for promoting keratinocyte culture. In this study, the expression of fibronectin and collagen-related factors, including integrins, by normal (NOK), SV40 T-antigen-immortalised (SVpgC2a) and malignant (SqCC/Y1) human oral keratinocytes, under standardised, serum-free conditions, was investigated by using microarray analysis. Cell growth was also studied in the presence and absence of a matrix consisting of human fibronectin and bovine collagen type I (FN–COL). Fibronectin transcripts were abundant in all cells, whereas 16 of 29 collagen chains and 14 of 24 integrin subunits were variably detected. With regard to both the expression level and the number of transcripts, higher collagen and lower integrin expression was observed in SVpgC2a cells than in NOKs and SqCC/Y1 cells. The cell types differed with regard to colony-forming efficiency and the rate and kinetics of growth at high cell density. For all cell types, FN–COL coating consistently stimulated cell migration, without influencing growth in mass culture or clonal density. The results demonstrate the transcription of genes associated with the formation and function of fibronectin and collagen in oral epithelium, and variably altered expression patterns in transformed states, and show that keratinocyte lines can be successfully transferred without the stimulus from extracellular FN–COL.
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| 36. |
- Scheers, Ellen M, et al.
(author)
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Cytotoxicity of amino alcohols to rat hepatoma-derived Fa32 cells.
- 2002
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In: ATLA (Alternatives to Laboratory Animals). - 0261-1929. ; 30:3, s. 309-12
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Journal article (peer-reviewed)abstract
- Amino alcohols are used as emulsifying agents in dry-cleaning soaps, wax removers, cosmetics, paints and insecticides. The cytotoxicities of 12 amino alcohols, which differed in chain length, position of the amino and alcohol groups, and the presence of an additional phenyl group, were determined by the neutral red uptake inhibition assay with normally cultured, glutathione-depleted or antioxidant-enriched Fa32 rat hepatoma-derived cells. Glutathione depletion and antioxidant enrichment were achieved by including 50(M L-buthionine-S,R-sulphoximine (BSO) or 100(M (-tocopherol acetate (vitamin E) in the culture medium for 24 hours before and during the assay. The cytotoxicity of the amino alcohols observed after treatment for 24 hours was expressed as the concentration of compound needed to induce a 50% reduction in neutral red uptake (NI50). The observed NI50 values ranged from 3mM to 30mM. The individual stereoisomers and a racemic mixture of 1-amino-2-propanol exhibited similar cytotoxicities (with normally cultured Fa32 cells, and vitamin E- and BSO-treated cultures). Similar NI50 values for D-(+)-2-amino-1-propanol, 3-amino-1-propanol and the L-, D- or DL- forms of 1-amino-2-propanol, indicated that the position of the amino group had little influence on the cytotoxicities of the amino alcohols. In contrast, the position of the hydroxyl group appeared to play an important role for the toxicity of the compound, as indicated by the significantly different NI50 values for 4-amino-1-butanol and 4-amino-2-butanol. An additional phenyl group greatly increased the cytotoxicity of 2-amino-1,3-propanediol. For most of the compounds, cytotoxicity increased when GSH was depleted, and decreased when the cells were enriched with vitamin E. This indicated that most of the tested chemicals interact with GSH, either directly or indirectly, by processes which generate oxygen free-radicals. Decreased toxicity was found for most of the chemicals administered to vitamin E-enriched cells, indicating that reactive oxygen species could be involved in the toxicity of the amino alcohols.
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| 37. |
- Staab, CA, et al.
(author)
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Modelling of normal and premalignant oral tissue by using the immortalised cell line, SVpgC2a: a review of the value of the model
- 2004
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In: Alternatives to laboratory animals : ATLA. - : SAGE Publications. - 0261-1929 .- 2632-3559. ; 32:4, s. 401-405
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Journal article (peer-reviewed)abstract
- Normal oral keratinocytes (NOKs), and a Simian virus 40 T-antigen-immortalised oral keratinocyte line termed SVpgC2a, were cultured in an effort to model the human oral epithelium in vitro, including normal and dysplastic tissue. Monolayer and organotypic cultures of NOKs and SVpgC2a were successfully established in a standardised serum-free medium with high levels of amino acids, by using regular tissue culture plastic for monolayers and collagen gels containing oral fibroblasts as the base for generating tissue equivalents. NOKs express many characteristics of normal tissue, including those associated with terminal squamous differentiation. After > 150 passages, SVpgC2a cells retained an immortal, non-tumourigenic phenotype that, relative to NOKs, was associated with aberrant morphology, enhanced proliferation, deficiency in terminal differentiation, proneness to apoptosis, and variably altered expression of structural epithelial markers. Transcript and protein profiling, as well as activity assays, demonstrated the expression of multiple xenobiotic-metabolising enzymes in SVpgC2a cells, some of which were higher in comparison to NOKs. A generally preserved, or even activated, ability for xenobiotic metabolism in long-term cultures of SVpgC2a cells indicated that this cell line could be useful in safety testing protocols — for example, in the development of consumer products in the oral health care field. However, SVpgC2a cells displayed some features reminiscent of a severe oral dysplasia, implying that this cell line could also to some extent serve as a model of a premalignant oral epithelium.
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| 38. |
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| 39. |
- Stigson, Michael, et al.
(author)
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Molecular targets and early response biomarkers for the prediction of developmental toxicity in vitro
- 2007
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In: ATLA (Alternatives to Laboratory Animals). - 0261-1929. ; 35:3, s. 335-342
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Journal article (peer-reviewed)abstract
- There is an urgent need for new in vitro methods to predict the potential developmental toxicity of candidate drugs in the early lead identification and optimisation process. This would lead to a reduction in the total number of animals required in full-scale developmental toxicology studies, and would improve the efficiency of drug development. However, suitable in vitro systems permitting robust high-throughput screening for this purpose, for the most part, remain to be designed. An understanding of the mechanisms involved in developmental toxicity may be essential for the validation of in vitro tests. Early response biomarkers - even a single one - could contribute to reducing assay time and facilitating automation. The use of toxicogenomics approaches to study in vitro and in vivo models in parallel may be a powerful tool in defining such mechanisms of action and the molecular targets of toxicity, and also for use in finding possible biomarkers of early response. Using valproic acid as a model substance, the use of DNA microarrays to identify teratogen-responsive genes in cell models is discussed. It is concluded that gene expression in P19 mouse embryocarcinoma cells represents a potentially suitable assay system, which could be readily used in a tiered testing system for developmental toxicity testing.
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| 40. |
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| 41. |
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| 42. |
- Weber, Tilo, et al.
(author)
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Case Studies Exemplifying the Transition to Animal Component-free Cell Culture
- 2022
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In: Alternatives to Laboratory Animals. - : SAGE Publications. - 0261-1929 .- 2632-3559. ; 50:5, s. 330-338
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Journal article (peer-reviewed)abstract
- Cell culture techniques are strongly connected with modern scientific laboratories and production facilities. Thus, choosing the most suitable medium for the cells involved is vital, not only directly to optimise cell viability but also indirectly to maximise the reliability of the experiments performed with the cells. Fetal bovine or calf serum (FBS or FCS, respectively) is the most commonly used cell culture medium supplement, providing various nutritional factors and macromolecules essential for cell growth. Yet, the use of FBS encompasses a number of disadvantages. Scientifically, one of the most severe disadvantages is the lot-to-lot variability of animal sera that hampers reproducibility. Therefore, transitioning from the use of these ill-defined, component-variable, inconsistent, xenogenic, ethically questionable and even potentially infectious media supplements, is key to achieving better data reproducibility and thus better science. To demonstrate that the transition to animal component-free cell culture is possible and achievable, we highlight three different scenarios and provide some case studies of each, namely: i) the adaptation of single cell lines to animal component-free culture conditions by the replacement of FBS and trypsin; ii) the adaptation of multicellular models to FBS-free conditions; and (iii) the replacement of FBS with human platelet lysate (hPL) for the generation of primary stem/stromal cell cultures for clinical purposes. By highlighting these examples, we aim to foster and support the global movement towards more consistent science and provide evidence that it is indeed possible to step out of the currently smouldering scientific reproducibility crisis.
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| 43. |
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