Troubleshooting tips for serum free media

So, you've moved your cells to a serum-free media and now they look bad. Been there 🙃 Here's what to check first, because your cells' disapproval might have nothing to do with the cell culture media formulation you've chosen.

1. Remove or reduce antibiotics by 5-10x

FBS binds and sequesters common cell culture antibiotics, so the activity level in culture is reduced compared to serum-free cultures. "1X" antibiotics in serum-free culture might impact your cells more than you'd expect.

2. Gentle passaging wins

FBS covers all manner of cell culture sins. Serum-free cultures often require gentler handling; minimise exposure to passaging reagents, keep cells and wash media warm, and try Accutase or TrypLE over trypsin. Trypsin also isn't inactivated by most serum-free media, so something to keep in mind!

3. Adaptation has an impact

Weaning cells gradually into a new media is gentlest on the cells and typically recommended. If using a direct adaptation process, move the cells into their new media when they aren't facing any other kind of passaging or thaw-related stress. For adherent cells, this means letting the cells attach to the plate and enter the exponential growth phase before performing a 100% feed to new media.

4. Adherent cells lifting up from the surface, looking like a viability issue? Rule out an adherence issue before playing with the media formulation.

Impaired attachment or spreading can very, very easily read as a viability issue, but requires a different trouble-shooting approach. Look for debris, check viability empirically, and consider eliminating FBS for the first time mid-passage, so (as much as possible) you can decouple the impacts of the media on viability/cell growth versus on attachment.

Not totally comprehensive, but the check list I wish I had when I started working in serum-free media dev ~6 years ago!