We will be updating this page with tips for best-practice and protocol considerations for achieving the best results.

An ongoing part of our lab work focusses on how we can make the gastruloid system reproducible and which factors are most important in achieving a “successful” culture (with axial elongations at >80% frequency). We can offer some general comments here:

Cell lines

When starting with a new cell line, we recommend that the initial cell number is titrated in a range around 200 cells/well in order to identify which condition gives optimal results. Our previous work has shown that the aggregate diameter at 48 hours after aggregation may be a good indicator of this property; we estimate that it should be around 150 µm ± 30 µm.

Culture media

In contrasting recent publications, it is evident that different groups maintain their ES cells under different conditions prior to making gastruloids. We think that this is an important consideration and one that should be kept in mind when interpreting their observations. We also anticipate some variation between groups that use commercially-available pre-mixed N2B27 and those that make it up from its constituent parts. While we routinely use the former, we advise users of “ready-made” N2B27 to be cautious in how this medium is stored and handled prior to use and would be happy to offer further tips in this regard.

Peter Baillie-Benson, June 2020