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:
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.
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
One additional question regarding the starting conditions that I’m wondering about: all protocols (J Vis Exp, Development, Biorxiv) describe the use of mESCs kept under ES+LIF (w/o feeders). However, cells kept for at least two passages tend to be a very heterogeneous population, with lots of cells already primed towards certain lineages. Also, culture of mESCs under ES+LIF w/o feeders is not the ‘typical’ EB starting condition. Have you tried starting with mESCs cultured under ES+LIF /w feeders? Or 2i+LIF as a starting condition?
Hi Jesse, thank you for your comments. We have never generated Gastruloids from mESCs that were cultured on feeders, however we have a number of experiments using cells cultured in 2i conditions. The responses of the Gastruloids following 2i culture are similar in a number of ways to what is obtained from ESL medium, however we have noticed some striking differences which we think is due to the differences in the heterogeneity of the system between cells cultured in 2i and ESL. Weâ€™re currently working on a manuscript which will present and discuss these results in much more detail, however, feel free to drop us an email (email@example.com or firstname.lastname@example.org) if you would like more details or whether youâ€™d like to discuss any experiments youâ€™ve done with the Gastruloids in the conditions youâ€™ve mentioned.