NC3R – CRACK it Challenge 36: Animal-free in vitro

Project duration: 2021–2021

Funding: National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs)

Principle investigators

• Prof. Dr. Henner Hollert
• Dr. Andreas Schiwy

Project partner

• Prof. Dr. Beate Escher (Helmholtz-Zentrum für Umweltforschung GmbH – UFZ)
• Dr. Rita Schlichting (Helmholtz-Zentrum für Umweltforschung GmbH – UFZ)
• Dr. Beat Thalmann (Scinora GmbH)
• Dr. Bart van der Burg and Dr. Peter Behnisch (BioDetection Systems b.v. (BDS)

Project webpage:   https://nc3rs.org.uk/

Project description

To improve in vitro toxicology animal derived products need to be substituted as they introduce variability and reduce the reproducibility. Especially, the cell culture component serum is of concern as it is harvested from foetal blood of calves and its composition is not defined and various production problems can impact its quality. It has been suggested that serum quality alone can be the cause of a reproducibility crisis. Nevertheless, important OECD test guidelines (TG) are still based on media containing serum. As requested in the CRACK IT Challenge 36 animal-derived components need to be substituted with a defined and reproducible alternative. A solution has been developed in context of the biopharmaceutical production with mammalian cell lines, which applies chemically defined media (CDM) since 1980s. In these media every component is defined, and its composition is reproducible. However, this development has been conducted only for a few cell lines like CHO cells and no OECD relevant cell lines. These media have been requested by the European Centre for the Validation of Alternative Methods (ECVAM) for the development of new cell-based methods and the OECD guidance document on “Good In vitro Method Practices (GIVIMP)" additionally states the importance of CDM. In the scientific community this problem has been addressed and a database for serum-free media has been established. One partner of our consortium, Scinora, develops and commercialises CDM for established cell lines (VERO, CHO, HROC) as well for the development of new cell lines (CHOsulu-ER). The GU has also experience with this technology in context of V79 cells and liver cell lines. For phase 1 of the challenge, we propose to adapt and to cultivate the relevant human cell lines for the OECD TGs 487 (A549) and 455 (ER CALUX®, ER-GeneBLAzer and CHOsulu-ER) in CDM.

In the context of effect-based methods the mammalian metabolism is an important element in in vitro toxicology that needs to be improved to achieve a realistic hazard assessment. This is of particular importance for compounds like Benzo[a]pyren (B[a]P) that show toxic effects after metabolic activation. In in vitro methods metabolic activation is simulated via animal-derived components like rat liver S9 or human organ donor derived S9. Again, these products, similar like serum, do not comply with 3R, are not defined and show high batch to batch variations. Here, we propose to substitute animal-derived products with a biotechnologically produced cocktail of metabolic enzymes called ewoS9. The biotechnological approach follows two principles: (1) The cultivation of cells in CDM, and (2) scaling of the process by cultivating the cells in suspension. The ewoS9 has been optimized for a high cytochrome P450 production through induction with selected P450 inducers. The activity of the cytochromes is monitored via biochemical assays. A quality criterion is the Cytochrome P450 CYP1A activity determined via the EROD assay. Following, the cells are harvested, processed and, like animal derived S9, the supernatant after centrifugation is obtained as the final product ewoS9R. This product has been evaluated within in vitro methods like the micronucleus assay with V79 and KCB H2B-eGFP cells. Furthermore, ewoS9 has been evaluated with the Ames fluctuation test according to IS0 1135011. Finally, we have investigated the metabolism of B[a]P after incubation with ewoS9R. The results showed the kinetics of B[a]P metabolism is comparable to rat derived S9. The advantage of ewoS9 as biotechnological produced cocktail of metabolic enzymes is the reduced variability.