April 13, 2018, updated April 16, 2018

t4 Workshop Report

Advanced Good Cell Culture Practice for human primary, stem cell-derived and organoid models as well as microphysiological systems

David Pamies, Anna Bal-Price, Christophe Chesné, Sandra Coecke, Andras Dinnyes, Chantra Eskes, Regina Grillari, Gerhard Gstraunthaler, Thomas Hartung, Paul Jennings, Marcel Leist, Ulrich Martin, Robert Passier, Jens C. Schwamborn, Glyn N. Stacey, Heidrun Ellinger-Ziegelbauer and Mardas Daneshian

doi:10.14573/altex.1710081

 

Summary
A major reason for the current reproducibility crisis in the life sciences is the poor implementation of quality control measures and reporting standards. Improvement is needed, especially regarding increasingly complex in vitro methods. Good Cell Culture Practice (GCCP) was an effort from 1996 to 2005 to develop such minimum quality standards also applicable in academia. This paper summarizes recent key developments in in vitro cell culture and addresses the issues resulting for GCCP, e.g. the development of induced pluripotent stem cells (iPSCs) and gene-edited cells. It further deals with human stem-cell-derived models and bioengineering of organo-typic cell cultures, including organoids, organ-on-chip and human-on-chip approaches. Commercial vendors and cell banks have made human primary cells more widely available over the last decade, increasing their use, but also requiring specific guidance as to GCCP. The characterization of cell culture systems including high-content imaging and high-throughput measurement technologies increasingly combined with more complex cell and tissue cultures represent a further challenge for GCCP. The increasing use of gene editing techniques to generate and modify in vitro culture models also requires discussion of its impact on GCCP. International (often varying) legislations and market forces originating from the commercialization of cell and tissue products and technologies are further impacting on the need for the use of GCCP. This report summarizes the recommendations of the second of two workshops, held in Germany in December 2015, aiming map the challenge and organize the process or developing a revised GCCP 2.0.

 

Keywords: quality control, cell culture, in vitro methods, gene editing, organotypic

 

------------------------------------------------------------------------------------------------------------------------------------------------------------

April 13, 2018; updated April 23, 2018

Research Article

A standardized method based on pigmented epidermal models evaluates sensitivity against UV-irradiation

Freia F. Schmid, Florian Groeber-Becker, Stefanie Schwab, Sibylle Thude, Matthias Goebeler, Heike Walles, Jan Hansmann

doi:10.14573/altex.1712211

 

Summary
To protect the human skin from extensive solar radiation, melanocytes produce melanin and disperse it via melanosomes to keratinocytes in the basal and suprabasal layers of the human epidermis. Moreover, melanocytes are associated with pathological skin conditions such as vitiligo and psoriasis. Thus, an in vitro skin model that comprises a defined cutaneous pigmentation system is highly relevant in cosmetic, pharmaceutical and medical research. Here, we describe how the epidermal-melanin-unit can be established in vitro. Therefore, primary human melanocytes are implemented in an open source reconstructed epidermis. Following 14 days at the air liquid interface, a differentiated epidermis was formed and melanocytes were located in the basal layer. The functionality of the epidermal-melanin-unit could be shown by the transfer of melanin to the surrounding keratinocytes, and a significantly increased melanin content of models stimulated with either UV-radiation or the melanin precursor dihydroxyphenylalanine. Additionally, an UV50 assay was developed to test the protective effect of melanin. In analogy to the IC50 value in risk assessment, the UV50 value facilitates a quantitative investigation of harmful effects of natural UV-radiation to the skin in vitro. Employing this test, we could demonstrate that the melanin content correlates with the resilience against simulated sunlight, which comprises 2.5 % UVB and 97.5 % UVA. Besides demonstrating the protective effect of melanin in vitro, the assay was used to determine the protective effect of a consumer product in a highly standardized setup.

 

Keywords: dermatology, reconstructed human epidermis, skin pigmentation, efficacy testing

------------------------------------------------------------------------------------------------------------------------------------------------------------

March 12, 2018

Research Article

Investigation of ruminant xenobiotic metabolism in a modified rumen simulation system (RUSITEC)

Barbara Birk, Alexander Stähle, Mathias Meier, Markus Palm, Dorothee Funk-Weyer, Gerhard Breves and Harald Seulberger

doi : 10.14573/altex.1712221

 

Summary

The approving agencies for plant protection agents request xenobiotic metabolism and residue studies in rats, farm animals and plants (e.g. EU regulation 1107/2009) according to OECD guidelines. The specific intestinal physiology of ruminants might lead to specific residues, which should be investigated very carefully. Specific aspects of xenobiotic metabolism in ruminants may arise, which are investigated by performing additional in vivo studies. The aim of the present work is to asses a modified rumen simulation system (RUSITEC) for such studies in vitro. Rumen constituents from sheep were incubated over 8 days. Physiological parameters followed by monitoring the pH (6.70 ± 0.07), the redox potential (301 mV ± 30 mV), the microbial composition and determination of β-glucosidase activity. After anequilibration period of three to four days the fermenters were probed with 14C-labelled triazolederivatives, i.e.common metabolites of azole fungicides. Only triazole-alanine was cleaved to 1,2,4-triazole, while triazole-acetic acid and triazole-lactic acid remained stable up to 96 h. Moreover, glycosides are often determined as the main residues in the plants. This analysis showed, that the two glucosides octyl-β-D-glucopyranoside and polydatin were both rapidly cleaved in this rumen in vitro system. These data showed that the modified RUSITEC system is stable, viable and maintained metabolic capacity over a longer period (at least 8 days). This makes many animal experiments obsolete and lead to a significant contribution of the 3R (refine, reduce, replace). The modification of the RUSITEC system enables safe routine use for unlabeled but also for radioactive labelled compounds.

 

Keywords: xenobiotic metabolism, livestock, RUSITEC, triazole derivative metabolites, glycosides

------------------------------------------------------------------------------------------------------------------------------------------------------------

 

March 11, 2018

Research Article

Testing vaginal irritation with the hen’s egg test-chorioallantoic membrane assay

Rita Palmeira-de-Oliveira, Rita Monteiro Machado, José Martinez-de-Oliveira and Ana Palmeira-de-Oliveira

doi : 10.14573/altex.1710091

 

Summary

The HET-CAM (Hen’s Egg Test-Chorioallantoic Membrane) assay is an in vitro alternative to the in vivo Draize Rabbit Eye test that mimics vascular changes in the chorioallantoic membrane. This qualitative method assesses the irritancy potential of chemicals. The CAM 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 potential for the eye and the vaginal epithelia is 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 corroborates clinical data, envisaging a possible in vitro/in vivo correlation. IS (B) was considered a better classification output. Although still requiring further validation, the HET-CAM assay seems an ideal prospect for vaginal irritancy potential in vitro studies.

 

Keywords: vaginal irritation, HET-CAM, in vitro

 

------------------------------------------------------------------------------------------------------------------------------------------------------------

 

February 23, 2018, updated February 24, 2018; updated March 5, 2018

Recommendation on Test Readiness Criteria for New Approach Methods in Toxicology: Exemplified for Developmental Neurotoxicity

Anna Bal-Price, Helena T. Hogberg, Kevin M. Crofton, Mardas Daneshian, Rex E. FitzGerald, Ellen Fritsche, Tuula Heinonen, Susanne Hougaard Bennekou, Stefanie Klima, Aldert H. Piersma, Magdalini Sachana, Timothy J. Shafer, Andrea Terron, Florianne Monnet-Tschudi, Barbara Viviani, Tanja Waldmann, Remco H.S. Westerink, Martin F. Wilks, Hilda Witters, Marie-Gabrielle Zurich and Marcel Leist

doi:10.14573/altex.1712081

Supplementary file (pdf)

 

Summary

Multiple non-animal-based test methods have never been formally validated. In order to use such new approach methods (NAMs) in a regulatory context, criteria to define their readiness are necessary. The field of developmental neurotoxicity (DNT) testing is used to exemplify the application of readiness criteria. The costs and number of untested chemicals are overwhelming for in vivo DNT testing. Thus, there is a need for inexpensive, high-throughput NAMs, to obtain initial information on potential hazards, and to allow prioritization for further testing. A background on the regulatory and scientific status of DNT testing is provided showing different types of test readiness levels, depending on the intended use of data from NAMs. Readiness criteria, compiled during a stakeholder workshop, uniting scientists from academia, industry and regulatory authorities are presented. An important step beyond the listing of criteria, was the suggestion for a preliminary scoring scheme. On this basis a (semi)-quantitative analysis process was assembled on test readiness of 17 NAMs with respect to various uses (e.g. prioritization/screening, risk assessment). The scoring results suggest that several assays are currently at high readiness levels. Therefore, suggestions are made on how DNT NAMs may be assembled into an integrated approach to testing and assessment (IATA). In parallel, the testing state in these assays was compiled for more than 1000 compounds. Finally, a vision is presented on how further NAM development may be guided by knowledge of signaling pathways necessary for brain development, DNT pathophysiology, and relevant adverse outcome pathways (AOP).


Keywords: developmental in vitro neurotoxicity testing, regulatory toxicology, toxicity screening, quality assurance

------------------------------------------------------------------------------------------------------------------------------------------------------------