Hi
We were transfecting HEK-293 cells adapted to grow in suspension to produce recombinant proteins with excellent results. Cells were maintained in Freestyle, transfected with PEI/DNA also in Freestyle, and fed after 48h with fresh medium plus tryptone. Supernatants were collected one week after the transfection to purify the proteins. Now our stock of Freestyle is exhausted. Do you know any other medium that could replace it? Any advice? We need a simple method that allows transfecting the cells at small scale with many different constructs and getting the proteins in a short time. The Fresstyle protocol is ideal, but whithout it....
This is an interesting question! Sadly, I can't provide an answer, but I have worked with the system as well and have thought about alternative media possibilities. Because let's be honest: the Freestyle medium is really, really expensive...
I have tried DMEM with and without FCS to no avail. The cells either die or clump really badly. Other manufacturers offer serum-free media for a bit less. Sigma offers the Ex-Cell media for example. I have not tried it yet.
I feel like the FCS is a big problem in HEK suspension culture. from my experience it causes the cells to clump. So maybe you could mix your own media with an FCS substitute? Perhaps use OptiMEM as a base and add serum-free supplement?
Good Luck! I'd be really happy if you'd reply if you find a suitable alternative!
Cheers,
Jan
Hi Gertrudis,
PEI transfection works in HEK 293 with DMEM + 5% FBS.
The question is if you want to totally avoid serum proteins and albumin in the sup whcih may interfere in protein purification, then you may have to look for other serum/protein- free medium.
However, if you only want to do expression analysis you may use this media condition.
Only, I seed the cells at 2.5X10e5 cell/ml (8 ml/100mm culture dish) concentration with DMEM+10% FBS overnight and then replace the media with DMEM+5% FBS before adding the PEI/DNA complex.
good luck,
Sudeep
My cells were adapted to grow in suspension at high densities and the goal is purifying the recombinant proteins. I could use serum (the lowest possible concentrations), but the media should be compatible with growth in suspension.
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