Abstract
Alginate hydrogels are a commonly used substrate for in vitro 3D cell culture. These naturally derived biomaterials are highly tunable, biocompatible, and can be designed to mimic the elastic modulus of the adult brain at 1% w/v solution. Recent studies show that the molecular weight of the alginate can affect cell viability and differentiation. The relationship between the molecular weight, viscosity and ratio of G:M monomers of alginate hydrogels is complex, and the balance between these factors must be carefully considered when deciding on a suitable alginate hydrogel for stem cell research. This study investigates the formation of embryoid bodies (EB) from mouse embryonic stem cells, using low molecular weight (LMW) and high molecular weight (HMW) alginates. The cells are differentiated using a retinoic acid-based protocol, and the resulting aggregates are sectioned and stained for the presence of stem cells and the three germ layers (endoderm, mesoderm, and ectoderm). The results highlight that aggregates within LMW and HMW alginate are true EBs, as demonstrated by positive staining for markers of the three germ layers. Using tubular alginate scaffolds, formed with an adapted gradient maker protocol, we also propose a novel 3D platform for the patterned differentiation of mESCs, based on gradients of retinoic acid produced in situ by lateral motor column (LMC) motor neurons. The end product of our platform will be of great interest as it can be further developed into a powerful model of neural tube development.