Commercially available synthetic hydrogels cannot replace Matrigel in promoting polarization and lumenogenesis of 3D ESC aggregates Authors

Pluripotency is defined as the ability of cells to differentiate into all cell types. In vivo, this capacity is gradually acquired by pluripotent cells in the epiblast as they polarize and arrange themselves into a three-dimensional structure known as a 'rosette', which then forms a lumen. Major epigenetic and metabolic changes occur during this crucial period of early development. Embryonic stem cells (ESCs), an in vitro model for early epiblast cells, can self-organise into rosettes when cultured in 3D, making them an ideal model for studying the events that accompany this morphogenesis. While this model is relevant to the 3Rs in terms of reducing the use of embryos, there is a significant drawback: the hydrogel commonly used for 3D cultures, Matrigel, is derived from animals. Furthermore, some rosette formation protocols use a serum-containing medium. The aim of this study was therefore to test alternative, commercially available, synthetic hydrogels and to compare the use of serum-containing and serumfree medium for modelling epiblast morphogenesis. Our results demonstrate that morphogenesis is more efficient in serum-free medium but did not occur in the synthetic hydrogels tested. This underscores the need for further optimisation of these systems.