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Transient Transfection: Adherent vs. Suspended/Floating Cells

expression adherent suspended transfection transient

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#1 chan8

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Posted 14 May 2012 - 08:26 AM

I am attempting to chemically transiently transfect HEK293 and CHO-K1 cells which are both adherent cell lines. The transgene protein expression that I am observing is lower than desired. Will changing to a floating or suspension cell line help increase protein epression? I have been told that floating cells may transfect at a higher rate. Also, I have heard that since suspension cells can be cultured a higher cell density than adherent cells, that the titre should increase with cell density (to a point). In your personal experience, have you found these things to be true?

Thank you.

#2 bob1

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Posted 14 May 2012 - 02:05 PM

Suspension cells, because they exist in a rounded state, have more surface area available for uptake of the transfection. You can do what is known as a reverse transfection, where you seed the cells at the same time as transfecting. This is typically more efficient for transfection, but does depend on what you are trying to transfect in (e.g. if you are transfecting something that interferes with attachment, then don't do it this way...), and on the cell type.

Titre increasing with cell density is true of pretty much any transfection!

#3 chan8

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Posted 14 May 2012 - 03:27 PM

Thanks bob1. This is what I suspected, but could never find any real-life examples or literature to back it up.

In regard to cell density, I knew there would be a difference, but to what extent I was unsure. Are adherent cells and suspension cells on completely different levels/scale in terms of protein production? I want to produce as much protein as possible (currently producing only 0.01-0.1 ug/ml), should I scrap the adherent cells?

#4 bob1

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Posted 14 May 2012 - 08:40 PM

For a given number of transfected cells, both will produce about the same amount of protein, it is purely that suspension cells can be grown in much larger numbers relatively easily if you have the right equipment.

If you need a lot of the protein for purification or something, have a think about producing it in bacteria, which pretty much guarantees you will get a lot of protein.

The amount of protein produced per cell varies a bit from cell line to cell line, but can also be influenced by the type of plasmid you are transfecting and the promoters you are using. Hopefully you are using a viral promoter (e.g. CMV or SV-40), which should give you high expression. If you have the right plasmid and cell line, the plasmid will be episomally maintained, rather than being slowly lost through cell division.

#5 chan8

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Posted 15 May 2012 - 08:51 AM

Thanks Bob. I considered using E. coli, but need precise folding.

Do non-primary mammalian cells like CHO-K1 and HEK293 (or their suspension adapted equivalents) express less protein as passage number increases or is that only with primary cells?

For a vector to be maintained episomally, does it require a viral element in the vector to allow it do so?

#6 bob1

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Posted 15 May 2012 - 03:22 PM

Non-primary lines do slowly loose expression as well, especially if you have kept the line up for more than about 10-15 passages. You can try fresh vials and/or transient transfections to enhance the expression.

For episomes, yes, you need the viral element and the respective viral integrants in the cell line too.

#7 chan8

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Posted 15 May 2012 - 03:30 PM

Are you saying that transient transfections are not affected by passages? I am transiently transfecting what I suspect are 30+ passage cells. Do passages not affect transient transfections as well?

#8 bob1

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Posted 16 May 2012 - 12:48 PM

Transient transfections are affected by passage number too, but in my experience, to a lesser extent - the DNA is still inserted and will express, whereas "stable" cell lines are often observed to lose the expression with increasing passage number.

You could also try in vitro transcription using rabbit reticulocyte lysate for expression of proteins. I don't know how mcuch protein you are likely to get out the end though.





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