mESC culturing - passage and morphology - (May/29/2008 )
Hi, everyone!
I'm quite new working with mouse ES cells and I've read some information about them in this forum. However, there are some issues that I'm still wondering.
For instance, how long (passages) can ES cells be maintained without differentiating? I'm using MEF cells as feeder in gelatin coated dishes. Well, I change medium and feeder every 1-2 days and I've tried once to culture ES cells to see how long they could last, but after the third passage, they already started to form big brown colonies, with a different morphology on the edges.
So, for how long can I culture them?
Thanks in advance.
I'm quite new working with mouse ES cells and I've read some information about them in this forum. However, there are some issues that I'm still wondering.
For instance, how long (passages) can ES cells be maintained without differentiating? I'm using MEF cells as feeder in gelatin coated dishes. Well, I change medium and feeder every 1-2 days and I've tried once to culture ES cells to see how long they could last, but after the third passage, they already started to form big brown colonies, with a different morphology on the edges.
So, for how long can I culture them?
Thanks in advance.
1. As long as they don't differentiate! But..
2. For making mouse evenetually, ideally 10 passage, but it may be +/- depending upon the ES cells you are using, and how you are treating them.
3. Try to finish your experiments within 3-4 passages, which means you will be freezing down a lot at each passage. Also depends upon at what passage you got the cells.
4. Do not continue culture when you don't need them for any experiments (i.e. just for maintaining them for future), that is a recipe for DISHaster.
5. Even if they don't differentiate, some will accumulate chromosomal aberrations (they do it fast), so less passage, better.
6. If you want to read some protocols and such, which might have some practical ideas, look up these links:
http://search.vadlo.com/b/q?sn=158621799&a...cells&rel=0
hth/
Thanks, cellcounter. I'll keep that in mind.
Oh, what if ES cells do not detach from each other? I've tried leaving in trypsin for 5 minutes, with ostensive mixing later, but it didn't work much. I'm worried about trypsinizing for a longer period, so I don't know if I should leave it longer.
Oh, what if ES cells do not detach from each other? I've tried leaving in trypsin for 5 minutes, with ostensive mixing later, but it didn't work much. I'm worried about trypsinizing for a longer period, so I don't know if I should leave it longer.
to break up the colonies, add medium after trypsin, and pipet up and down in the plate pushing (gently) the pipet on the plate surface, this should provide enough friction to brake them up in single cells. Alternatively you can use a P1000 to pipet them up and down, this will brake them better but it's a bit harsh too (more die during passaging).
Thank you, cmonet!
I've tried what you said, but still, a lot of colonies remain. Now I am trying using a higher concentration of trypsin, for a bit longer, too. So far, it seems to be working.
But I still have to keep training it.
Thanks a lot.