Subculturing cells grown in serum free media - (Jul/20/2005 )

Hello everyone,

I am currently growing HMEC, MCF-10A and MCF-12A in MEGM, serum-free media. I got them in flasks and have cultured them to confluence. However, I subcultured them a few days ago, and until today, the cells don't seem to be fully adherent and spreading out on the flask. They still appear round and don't seem to be multiplying.

Here is what I did, could anyone comment if I'm doing anything wrong, or suggest a better method?

Working on a confluent T25 flask.

1. Thaw 3 ml MEGM to 37C
2. Thaw 1 ml trypsin-EDTA (from Cambrex, Clonetics) to room temp
3. Thaw 2 ml trypsin neutralising solution (from Cambrex, Clonetics) to room temp

4. Aspirate old media
5. Rinse monolayer with PBS
6. Aspirate PBS
7. Add 1 ml trypsin, wait 5 minutes till cells are rounded up
8. Add 2 ml trypsin neutralising solution
9. Add 3 ml MEGM
10. Split 1:2, ie. 3 ml cell suspension + 12 ml fresh media in a T75 flask
11. Incubate at 37C, 5% CO2

Look forward to some advice.

Many thanks!

serum free media starves the cells. this puts them in g0, and they won't go into the g1 phase until they're feed.
i follow a very similar method with my MCF-7s, but after i've subcultured them, i plate them in normal media for a few hours, or overnight, until they stick down, and then wash the wells in PBS, and then use the serum free media.
hope that helps,
not sure how 'right' it is, but it works.

Many thanks Vetticus!

Think I will do what you suggested. Probably feed them with 90% media and 10% FBS today, and change to serum-free media tomorrow.

Hope it works.