An Optimized Protocol for the Extended Culture of Gastruloids from Mouse Embryonic Stem Cells

Gastruloids are three-dimensional aggregates, derived from mouse embryonic stem cells, that recapitulate key events of early embryogenesis. Protocols to generate gastruloids are highly sensitive to aggregation conditions, often resulting in variability. Here, we present an optimized method for generating gastruloids and extending their culture up to 168 h post-aggregation by embedding them in 10% Matrigel at 96 h. This protocol enables reproducible generation of gastruloids with derivatives of all three germ layers, providing an extended experimental window to study post-gastrulation developmental processes in vitro.