In vitro embryolethality testing during the peri-implantation stage using 3D mouse embryoids: Comparison with 2D mouse cell cultures and the zebrafish embryo model

The recent development of in vitro embryoid models may be a way to reduce or even replace animals in developmental and reproductive toxicity (DART) studies. Here, we evaluated an in vitro mouse 3D embryoid model using three stem cell lineages including mouse embryonic stem cells (ESC), trophoblast stem cells (TSC) and extra-embryonic endoderm stem cells (XENC) to detect embryolethality of seven known positive or negative compounds at the peri-implantation stage in vivo. We performed a comparative study by testing these substances in parallel on monocultures of the individual cell lines in 2D and in zebrafish embryos. Embryoids were exposed to each compound for 48 h and analysed on day in vitro (DIV) 4, which resemble the peri-implantation embryo at E5.5 in vivo. Metabolic activity, morphological development and gene expression were evaluated. We showed that all early direct embryolethal compounds significantly reduced the metabolic activity of the embryoids at an in vivo relevant concentration range. Furthermore, this model could also detect embryolethal compounds that have specific adverse effects on extraembryonic structures. The additional endpoints (gene expression or morphological assessments) were required to detect adverse effects when only a minoritarian cell type of the embryoid was affected. In contrast, zebrafish embryos had a very low sensitivity, and the monocultures of the individual cell lines were less specific than the 3D embryoids. Combining 2D and 3D testing with carefully selected additional endpoints could serve as a powerful tool to assess the potential effects of new chemicals on embryo development at the peri-implantation stage.