Hi, our lab normally uses feeder-free method to maintain E14TG2a, mES cell line. Recently, I am trying to thaw the ATCC E14TG2a new cell line, which recommends to be grown on feeder cell. Many companies provide primary MEF, which can go for 5 passages then crisis will happen. So many labs use some ways to immortalise MEF to make it a permanent cell line. Our lab now has immortalised MEF P4-P5, can I use them as feeder cell for E14TG2a?
For those experienced labs using feeder cell, do you always use primary MEF? Do you need to freeze some at P2-P3, then use them as feeder cell before P5?
Thanks so much!
Feeder cells are usually made as a large irradiated stock. The irradiation serves to mitotically inactivate feeder cells so they don't outgrow your ES cells. Here is an example of a protocol: https://genetics.emory.edu/documents/labs/caspary/FEEDERPREPGAMMA31AE9B.pdf
Here is a protocol from CSH: doi:10.1101/pdb.prot4400
When I was making fetal feeders from 13.5 dpc pups, each pup was eviscerated and the head was cut off. After dissociation, the number of pups matched the number of primary 100mm plates they went into. Upon 70% confluence they were frozen one plate per vial. As needed, each vial was thawed into 5 100mm plates at P1. These were split 2 more times at the same ratio to yield 125 100mm plates at P3. These were dissociated and irradiated and plated or frozen at a cell count of 1.8 x 10e6 per plate. Thus one pup usually was good for over 300 plates.
I tried going one additional passage to P4 but the quality of the feeders was dramatically reduced. In my opinion, it's just not worth it. Especially given how easy it is to make a large stock of high quality feeders with relatively few mice. I've never seen commercial feeders that looked remotely as good as the ones I easily made myself.
All that said, this immortalized line might be just fine with the additional few passages.
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