What day after EB Day 0 do I add iMSC Expansion Media and what day do I add iMSC Maintenance Media, exactly? Also how are you replating?

Dear Cristina,

Thank you for contact us about our protocol. I hope that the answers are clear and concise. Please. Let me know if you have any additional questions or is needed more clarification. Best Regards.

Q1)

If EB Day 0 is when I place the 15K cell droplets using the hanging drop method, is EB Day 2 or EB Day 3 when I change media to iMSC Expansion media?

Answer:

On EB Day 3, transfer the EBs onto 1% gelatin–coated plates and replace mTeSR-1 with iMSC Expansion Medium supplemented with VEGF and BMP4. Day 3 of EB formation corresponds to Day 0 + 2 days = 3 days. iPSC and ESC tend to form spontaneous aggregates, which are already visible a few hours after the cell suspension (key step).

Q2)

And what day after EB Day 0, do I change to iMSC Maintenance media?

Answer:

After 3 days in Expansion Medium (i.e., typically EB Day 6), you will notice that EBs begin attaching to the gelatin-coated plates, while some may still be floating. At this stage, replace the medium with iMSC Maintenance Medium and continue culture until the majority of EBs are attached (another 5–7 days). Eventually, the flattened cells will expand to confluence. Do not passage until the plate is at least 60% confluent (can take sevral days).

Q3)

When first replating the emerging MSC precursors, what is the cell density at which they are replated onto gelatin-coated plates?

Answer:

At the first passage (after the outgrowth becomes 80% confluent), dissociate using Accutase–PBS (1:1 dilution). Replate the cells, without reaching the dissociation to single cells, (it is possible to see chunks of the EB after passaging, this supports survival rather than early selection). The protocol specifies transferring to two 1% gelatin coated dishes rather than seeding at a defined density (this is depending of the initial density of the appearing flat cells (if the flat cells are in a 80% density, I will plate in 2 dishes, otherwise I will recommend only one dish). This is labeled as Passage 0 (P0). This is a key step: overly harsh dissociation can damage the emerging iMSC. In subsequent passages, the iMSC population will be progressively selected by the media and culture conditions and can be counted.