Optimization of Gastruloid Pre-culture Conditions for Reproducible Germ Layer Composition

Gastruloids are an in vitro model that mimics key aspects of gastrulation and can be used to study implantation-stage embryonic development. Even though current protocols have contributed significantly to our understanding of developmental biology, further improvements in terms of consistency and reproducibility are still required. In this chapter, we present a detailed pre-culture protocol optimized for the generation of gastruloids from 129S1/SvImJ/ C57BL/6 mouse embryonic stem cells (mESCs). Additionally, we provide information on the steps of the protocol open to optimization and present a workflow for selecting optimal conditions and evaluating the gastruloid formation outcome when using different cell lines. Overall, this workflow facilitates a step-by-step approach toward generating gastruloids from any cell line in a highly reproducible manner by optimizing the mESC pre-culture conditions.