Embryonic stem cell culture - (Sep/23/2007 )

Hi everyone,

I am new of doing embryonic stem cell culture. I see some information about culturing embryonic stem cell that there should be a feeder layer for inhibiting cell differentiation. So when I subculture the cells using trypsin, will feeder layer cells also be loosen and coolected together with the embryonic stem cells?

In addition, can I use gelatin instead of feeder layer cells?

Thanks for your attention!

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Ryan
As you know trypsin acts on proteins at their carboxyl terminus of aromatic amino acids. And therefore the adhesion proteins will be proteolysed and it will affect contamination of feeder layer with stem cells. Gelatin is formed from collagen boiling and cooling and it contains proline-hydroxyproline-glycine and also due to inaccessibility of trypsin to gelatin network, it will not or little affect the gelatin structure and integrity.
Ajay

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You don't use trypsin on stem cells that you are culturing for production. What do you mean by subculture?

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production of? mice? I do use trypsin, no problem.
Some ES cell lines require the feeders layer to remain undifferentiated, some others don't, this will be depentend on the cell line you're using. Yes you will resuspend the feeders too with trypsin, but generally you will need to subculture them on a plate with fresh new feeders since during passage you typically end up diluting them 1:3-1:6 so you would dilute the feeders too. The feeders are (or should be) mytotically inactivated so they dont proliferate like the ES cells and you'd loose them during passaging if you dont plate on fresh ones.

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It’s very easy ryan, you just transferred your trypsinized cells to a non coated flask ,then you should put them in the incubator at 37 ºC for 45 minutes. After this time, collect the medium and you will have your stem cells without (or practically without) feeders.

Jose

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