ALTEX - Alternatives to animal experimentation https://www.altex.org/index.php/altex <p><strong>The journal ALTEX – Alternatives to Animal Experimentation publishes open access academic articles on the development and implementation of alternatives to the use of animals for scientific purposes and informs on international developments in this field. </strong></p> <p>ALTEX is the official organ of&nbsp;<a href="http://caat.jhsph.edu/" target="_blank" rel="noopener">CAAT</a>, <a href="https://www.biologie.uni-konstanz.de/leist/caat-europe/">CAAT-Europe</a>, the Doerenkamp-Zbinden Chairs, <a href="https://www.eusaat-congress.eu/index.php/eusaat">EUSAAT</a> and <a href="http://altweb.jhsph.edu/about_us/t4.html">t<sup>4</sup></a>.</p> ALTEX Edition / Springer Spektrum Springer-Verlag GmbH en-US ALTEX - Alternatives to animal experimentation 1868-596X <p>Articles are distributed under the terms of the Creative Commons Attribution 4.0 International license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is appropriately cited (CC-BY). Copyright on any article in ALTEX is retained by the author(s).</p> Editorial https://www.altex.org/index.php/altex/article/view/1186 Sonja von Aulock ##submission.copyrightStatement## http://creativecommons.org/licenses/by/4.0/ 2018-10-24 2018-10-24 35 4 U2 U2 Sex and media: Considerations for cell culture studies https://www.altex.org/index.php/altex/article/view/1028 <p>Cell culture has enhanced our understanding of cellular physiology and constitutes an important tool in advancing mechanistic insight. Researchers should be reminded, however, that there are limitations in extrapolating data derived from cultured cells to questions focusing on the impact of sex. In this <em>Opinion</em>, we highlight two underappreciated aspects of cell culture systems regarding sex: how cell culture media alters the sex hormone environment, and how the innate sex of the cell is often not factored into the overall analysis. By paying careful attention to these areas, researchers can facilitate reproducibility of their cell culture models, which is consistent with the mandate from the National Institutes of Health to improve scientific rigor and reproducibility in research.</p> Roberta de Souza Santos Aaron P. Frank Biff F. Palmer Deborah J. Clegg ##submission.copyrightStatement## http://creativecommons.org/licenses/by/4.0 2018-10-24 2018-10-24 35 4 435 440 10.14573/altex.1806151 A human population-based organotypic in vitro model for cardiotoxicity screening https://www.altex.org/index.php/altex/article/view/1024 <p>Assessing inter-individual variability in responses to xenobiotics remains a substantial challenge, both in drug development with respect to pharmaceuticals and in public health with respect to environmental chemicals.&nbsp; Although approaches exist to characterize pharmacokinetic variability, there are no methods to routinely address pharmacodynamic variability.&nbsp; In this study, we aimed to demonstrate the feasibility of characterizing inter-individual variability in a human <em>in vitro </em>model. Specifically, we hypothesized that genetic variability across a population of iPSC-derived cardiomyocytes translates into reproducible variability in both baseline phenotypes and drug responses. We measured baseline and drug-related effects in iPSC-derived cardiomyocytes from 27 healthy donors on kinetic Ca<sup>2+</sup> flux and high-content live cell imaging.&nbsp; Cells were treated in concentration-response with cardiotoxic drugs: isoproterenol (β-adrenergic receptor agonist/positive inotrope), propranolol (β-adrenergic receptor antagonist/negative inotrope), and cisapride (hERG channel inhibitor/QT prolongation). &nbsp;Cells from four of the 27 donors were further evaluated in terms of baseline and treatment-related gene expression. Reproducibility of phenotypic responses was evaluated across batches and time. iPSC-derived cardiomyocytes exhibited reproducible donor-specific differences in baseline function and drug-induced effects. We demonstrate the feasibility of using a panel of population-based organotypic cells from healthy donors as an animal replacement experimental model. This model can be used to rapidly screen drugs and chemicals for inter-individual variability in cardiotoxicity. This approach demonstrates the feasibility of quantifying inter-individual variability in xenobiotic responses, and can be expanded to other cell types for which <em>in vitro</em> populations can be derived from iPSCs. &nbsp;</p> Fabian Grimm Alexander Blanchette John S. House Kyle Ferguson Nan-Hung Hsieh Chimeddulam Dalaijamts Alec A. Wright Blake Anson Fred A. Wright Weihsueh A. Chiu Ivan Rusyn ##submission.copyrightStatement## http://creativecommons.org/licenses/by/4.0 2018-10-24 2018-10-24 35 4 441 452 10.14573/altex.1805301 Material-mediated pyrogens in medical devices: Applicability of the in vitro Monocyte Activation Test https://www.altex.org/index.php/altex/article/view/1027 <p>Pyrogenicity presents a challenge to clinicians, medical device manufacturers, and regulators. A febrile response may be caused by endotoxin contamination, microbial components other than endotoxin, or chemical agents that generate a material-mediated pyrogenic response. While test methods for the assessment of endotoxin contamination and some microbial components other than endotoxin are well-established, material-mediated pyrogens remain elusively undefined. This review presents the findings of literature searches conducted to identify material-mediated pyrogens associated with medical devices. The <em>in vivo </em>rabbit pyrogen test (RPT) is considered to be the “gold standard” for medical device pyrogenicity testing, despite the fact that few medical device-derived material-mediated pyrogens are known. In line with global efforts to reduce the use of research animals, an <em>in vitro </em>monocyte activation test (MAT) has the potential to replace the RPT. The MAT is used to detect substances that activate human monocytes to release cytokines. This review will also describe the potential opportunities and challenges associated with MAT adoption for the detection of material-mediated pyrogens in medical device testing.</p> Lindsey K. Borton Kelly P. Coleman ##submission.copyrightStatement## http://creativecommons.org/licenses/by/4.0 2018-10-24 2018-10-24 35 4 453 463 10.14573/altex.1709221 Completely defined co-culture of adipogenic differentiated ASCs and microvascular endothelial cells https://www.altex.org/index.php/altex/article/view/620 <p>Vascularized adipose tissue models are in high demand as alternatives to animal models to elucidate the mechanisms of widespread diseases, screen for new drugs or assess drug safety levels. Animal-derived sera such as fetal bovine serum (FBS), which are commonly used in these models, are associated with ethical concerns, risk of contaminations and inconsistencies of their composition and impact on cells. In this study, we developed a serum-free, defined co-culture medium and implemented it in an adipocyte/endothelial cell (EC) co-culture model.<br>Human adipose-derived stem cells were differentiated under defined conditions (diffASCs) and, like human microvas­cular ECs (mvECs), cultured in a defined co-culture medium in mono-, indirect or direct co-culture for 14 days. The defined co-culture medium was superior when compared to mono-culture media and facilitated the functional mainte­nance and maturation of diffASCs including perilipin A expression, lipid accumulation, and also glycerol and leptin release. The medium also allowed mvEC maintenance, confirmed by the expression of CD31 and von Willebrand factor (vWF), and by acetylated low-density lipoprotein (acLDL) uptake. Thereby, mvECs showed strong dependence on EC-specific factors. Additionally, mvECs formed vascular structures in direct co-culture with diffASCs.<br>The completely defined co-culture system allows for the serum-free culture of adipocyte/EC co-cultures and thereby represents a valuable and ethically acceptable tool for the culture and study of vascularized adipose tissue models.</p> Ann-Cathrin Volz Larissa Hack Franziska B. Atzinger Petra J. Kluger ##submission.copyrightStatement## http://creativecommons.org/licenses/by/4.0 2018-10-24 2018-10-24 35 4 464 476 10.14573/altex.1802191 Adaptation of the human Cell Line Activation Test (h-CLAT) to animal-product-free conditions https://www.altex.org/index.php/altex/article/view/1026 <p>Skin sensitizers are substances that can elicit allergic responses following skin contact. The process by which this occurs, i.e., skin sensitization, is a series of key events that form an adverse outcome pathway (AOP). Key event 3 in the AOP is dendritic cell activation that can be modelled by the human Cell Line Activation Test (h-CLAT) and is typified by changes in cell surface markers CD54 and CD86 in dendritic cells. The h-CLAT is accepted at a regulatory level (OECD Test Guideline 442E) and can be used to assess skin sensitization potential as part of an integrated approach to testing and assessment (IATA).</p> <p>Stakeholders in the cosmetics and chemical industries have scientific and ethical concerns relating to use of animal-de­rived material and have communicated a strong preference for fully human-based <em>in vitro </em>methods. Therefore, we adapted the h-CLAT to animal-product-free conditions and validated the adapted method with the proficiency panel substances listed in Annex II of TG 442E using 3 independent batches of pooled human serum. The modified method showed equivalence to the validated reference method (VRM), as all proficiency substances were correctly classified. Comparable values for CV75 (concentration yielding 75% cell viability), EC150 and EC200 (concentration yielding RFI of ≥ 150 for CD86 and ≥ 200 for CD54) were obtained. Data generated using the adapted method may be used in European REACH submissions, provided the proficiency data is included. We are seeking formal inclusion of the adaptation into TG 442E, enabling compliance with global regulations.</p> Alexander Edwards Lottie Roscoe Christopher Longmore Fiona Bailey Bushra Sim Carol Treasure ##submission.copyrightStatement## http://creativecommons.org/licenses/by/4.0 2018-10-24 2018-10-24 35 4 477 488 10.14573/altex.1710051 Replacement of foot-and-mouth disease virus cattle tongue titration by in vitro titration https://www.altex.org/index.php/altex/article/view/1140 <p>Titration of foot-and-mouth disease cattle challenge virus in cattle tongue has been the standard for many years in many countries, although titration in animals has been replaced by <em>in vitro </em>methods for all other applications. The objective of the analysis was the replacement of <em>in vivo </em>titration of cattle challenge virus by <em>in vitro </em>titration. Using data from 32 <em>in vivo </em>titration experiments together with the <em>in vitro </em>titration results of the same samples obtained by plaque count on primary lamb or pig kidney cells, as well as data from the virus isolation control chart used in the laboratory, we show that the reproducibility of the <em>in vitro </em>titration is much higher than that of the <em>in vivo </em>titration. The titer on primary kidney cells was on average 1.4 log<sub>10</sub> higher than the titer determined by titration in cattle tongue (PFU/ml compared to bovine ID<sub>50</sub>/ml), but the difference varied among different strains. The study also shows that the probability of infection in cattle tongue is high even when a lower challenge dose is used, which makes the variability between strains less important. Based on these results, we propose to change the standard dose for cattle challenge from 10<sup>4</sup> bovine ID<sub>50</sub> to 10<sup>5.4</sup> PFU, and to replace the <em>in vivo </em>cattle tongue titration method with the <em>in vitro </em>titration method.</p> Aldo Dekker Froukje van Hemert-Kluitenberg Anna H. Oosterbaan Kimberly Moonen Laure Mouton ##submission.copyrightStatement## http://creativecommons.org/licenses/by/4.0 2018-10-24 2018-10-24 35 4 489 494 10.14573/altex.1712222 Testing vaginal irritation with the Hen’s Egg Test-Chorioallantoic Membrane assay https://www.altex.org/index.php/altex/article/view/552 <p>The HET-CAM (Hen’s Egg Test-Chorioallantoic Membrane) assay is an <em>in vitro </em>alternative to the <em>in vivo </em>Draize rabbit eye test. This qualitative method assesses the irritancy potential of chemicals. The chorioallantoic membrane responds to injury with an inflammatory process similar to that in the rabbit eye’s conjunctival tissue. Regarding topical toxicity assessment of medical devices, ISO 10993-10 states that any skin or eye irritant material shall be directly labelled as a potential vaginal irritant without animal testing, suggesting that the irri­tation potentials for the eye and the vaginal epithelia are similar. The aim of this work was to apply the HET-CAM assay to test the irritancy potential of vaginal formulations. Vaginal semisolid medicines and lubricants currently marketed were tested along with the Universal Placebo formulation that has been shown to be clinically safe. Nonoxynol-9 (N-9), a known vaginal irritant, was enrolled as positive control (concentrations ranging from 0.001 to 100% (v/v)). The assay was conducted according to the ICCVAM – Recommended Test Method (NIH Publication No. 10-7553 – 2010). Formulations were then classified according to irritation score (IS), using the analysis methods (A) and (B). The studied vaginal formulations showed low potential for irritation. N-9 was classified as a severe irritant at concentrations above 2%, which is in line with clinical data, envisaging a possible <em>in vitro/in vivo </em>correlation. IS (B) was considered a more detailed classification output. Although still requiring further validation, the HET-CAM assay seems an ideal prospect for <em>in vitro </em>vaginal irritancy testing.</p> Rita Palmeira-de-Oliveira Rita Monteiro Machado José Martinez-de-Oliveira Ana Palmeira-de-Oliveira ##submission.copyrightStatement## http://creativecommons.org/licenses/by/4.0 2018-10-24 2018-10-24 35 4 495 503 10.14573/altex.1710091 Reevaluating the role of megalin in renal vitamin D homeostasis using a human cell-derived microphysiological system https://www.altex.org/index.php/altex/article/view/729 <p>The role of megalin in the regulation of renal vitamin D homeostasis has previously been evaluated in megalin-knockout mice and rat proximal tubule epithelial cells. We revisited these hypotheses that were previously tested solely in rodent models, this time using a 3-dimensional proximal tubule microphysiological system incorporating primary human proximal tubule epithelial cells. Using this human cell-derived model, we confirmed that 25OHD3 is transported into the human proximal tubule epithelium via megalin-mediated endocytosis while bound to vitamin D binding protein. Building upon these findings, we then evaluated the role of megalin in modulating the cellular uptake and biological activity of 1α,25(OH)2D3. Inhibition of megalin function decreased the 1α,25(OH)2D3-mediated induction of both cytochrome P450 24A1 protein levels and 24-hydroxylation activity following perfusion with vitamin D binding protein and 1α,25(OH)2D3. The potential for reciprocal effects from 1α,25(OH)2D3 on megalin expression were also tested. Contrary to previously published observations from rat proximal tubule epithelial cells, 1α,25(OH)2D3 did not induce megalin gene expression, thus highlighting the potential for meaningful interspecies differences in the homeostatic regulation of megalin in rodents and humans. These findings challenge a recently promoted hypothesis, predicated on the rodent cell data, that attempts to connect 1α,25(OH)2D3-mediated regulation of renal megalin expression and the pathology of chronic kidney disease in humans. In addition to providing specific insights related to the importance of renal megalin in vitamin D homeostasis, these results constitute a proof-of-concept that human-derived microphysio­logical systems are a suitable replacement for animal models for quantitative pharmacology and physiology research.</p> Brian D. Chapron Alenka Chapron Brian Phillips Miracle C. Okoli Danny D. Shen Edward J. Kelly Jonathan Himmelfarb Kenneth E. Thummel ##submission.copyrightStatement## http://creativecommons.org/licenses/by/4.0 2018-10-24 2018-10-24 35 4 504 515 10.14573/altex.1803161 Lowering the p-value from 0.05 to 0.005 conflicts with the 3R rules – an advocacy for alternatives to hypothesis testing with the p-value approach https://www.altex.org/index.php/altex/article/view/1137 Konradin Metze Fernanda Aparecida Borges da Silva Irene Lorand-Metze ##submission.copyrightStatement## http://creativecommons.org/licenses/by/4.0 2018-10-24 2018-10-24 35 4 516 517 10.14573/altex.1807091 The ethical evaluation of animal experiments deserves more than empty phrases https://www.altex.org/index.php/altex/article/view/1172 Tilo Weber ##submission.copyrightStatement## http://creativecommons.org/licenses/by/4.0 2018-10-24 2018-10-24 35 4 518 519 10.14573/altex.1810041 3Replacement Winter School – Out of the barriers: In vitro models in toxicology https://www.altex.org/index.php/altex/article/view/1143 Francesca Caloni Yula Sambuy Guerino Lombardi Silvia Dotti Isabella De Angelis ##submission.copyrightStatement## http://creativecommons.org/licenses/by/4.0 2018-10-24 2018-10-24 35 4 520 521 10.14573/altex.1807231 Corners https://www.altex.org/index.php/altex/article/view/1174 Sonja von Aulock ##submission.copyrightStatement## http://creativecommons.org/licenses/by/4.0 2018-10-24 2018-10-24 35 4 522 526 ALTEX Edition membership application https://www.altex.org/index.php/altex/article/view/1187 Sonja von Aulock ##submission.copyrightStatement## http://creativecommons.org/licenses/by/4.0/ 2018-10-24 2018-10-24 35 4 527 527 Imprint https://www.altex.org/index.php/altex/article/view/1188 Sonja von Aulock ##submission.copyrightStatement## http://creativecommons.org/licenses/by/4.0/ 2018-10-24 2018-10-24 35 4 528 528 TIERethik https://www.altex.org/index.php/altex/article/view/1189 Petra Mayr ##submission.copyrightStatement## http://creativecommons.org/licenses/by/4.0/ 2018-10-24 2018-10-24 35 4 U3 U3 Cover https://www.altex.org/index.php/altex/article/view/1191 Sonja von Aulock ##submission.copyrightStatement## http://creativecommons.org/licenses/by/4.0/ 2018-10-24 2018-10-24 35 4