It is fairly simple to turn CHO/CHO Lec cells (and Hek293) into suspension culture. We use chemically defined serum free medium (e.g.CHO Pro293S-CDM or ProCHO 4 Protein-free Medium from Lonza). Adherent grown cells will be harvested by trypsin procedure. Then cells will be seeded in a mixture of regular medium / chemical defined medium (50:50) and cultured for 3 days. Next cells will be harvested and reseeded in a mixture of regular medium / chemical defined medium (25:75) and cultured for 3 days. Thereafter cells will be harvested and reseeded in a pure chemical defined medium and cultured for 3 days. Now cells should grow in suspension. You may test cell viability etc. to ensure the quality of suspension cells.

alpha MEM + 10% FCS (no need to heat inactivate FCS), rotate @ 37C at ~ 50 rpm, don't go below 4E4 C/ml, not above 1.2E6 C/ml. CHO aren't good expressors, though. They typically express about 100 times less than Sf9 cells w/ Baculo in mg product/mg protein. You want to select a high expressing clone first, then move into suspension. In Industry, they subclone the clone before fermentation. There are vectors that help you express higher (nowhere near baculo, but at least o.k.) levels with sorting (w/ IRES EGFP) but populations aren't stable. You can do limited dilution in DMEM 10% FCS to clone. Transient transfection is done w/ adherent cells typically. Discard cells that are older than 5 months since they change a few lectin binding properties.

Thank you for you answer.

I know that the baculovirus system gives better yields, but the glycosylations are a problem. I already have a good yield in CHO cells but in roller bottles and I would like to try switching to suspension.

So do you just trypsinize the adherent cells and transfer them to the media you are talking about?

Exactly, that's how it's done. Obviously, avoid clumping. alpha MEM doesn't have enough Mg2+ for the cells to reattach. Give them some time to adapt (~ 4 days?). I'd go in with at least 5E5 C/ml. Which Lecs are you using?

I'm using the 3.2.8.1 cells. Is there a way to adapt them to serum-free media as well?

Hi Goran, literature describes several procedures involving step-wise reduction of serum in the medium to 0% which will eventually lead to some cells de-taching and starting to grow in suspension. Interestingly, I have made good experience with the less subtle approach to trypsinize your cells and re-seed them into the same growth medium but without serum into a shake flasks at >5E05 cells/ml.

Then just wait. Change medium from time to time (maybe every 4-5 days). The cells will first not proliferate and viability can drop to ~60-70%. But they will recover and start proliferating again in suspension after a while. It can take 10-18 days.

Good luck!

Hi, I see you got already some good answers.

Here are two useful sites:

1) a protocol for the adaptation you are looking for:

http://www.lifetechnologies.com/us/en/home/references/protocols/cell-culture/chinese-hamster-ovary-cell-culture-protocol/transferring-cho-cells-from-monolayer-to-suspension-culture.html

2) an example protocol for handling CHO suspension strains.

http://bio.lonza.com/fileadmin/groups/marketing/Downloads/Protocols/Generated/Optimized_Protocol_111.pdf

Note: As you likely are well aware, there are already CHO strains which are maintained in suspension. Whether you use one of them or create your own, you need to be aware that you will have a different cell from what you have now in adherent culture. As long as you just want high yield of some over-expressed protein, you will likely be fine. For more mechanistic studies, you will need additional controls to characterize the difference between the lines, they can be quite significant.

(I don't work for either of the companies I linked to :-)

Good luck!

Anka

Once they are growing well, you can keep them at 5% FCS, though they don't grow as fast, then. (I'd plan an experiment for about day 8 day after transfer when it was going well.) Personally, I never had them FCS free because it shouldn't make a difference for the glycosylation pattern (the transferase and transporter for Sia, as well as GnT-I are k.o.'d, the Golgi transporter for Gal as well) while you'll likely experience another decrease in production. I think I faintly remember people using serum free media with the parent cell line at least (Pro5), so that should be possible, although some Lecs might not at all like it. Don't remember which medium they took.

We have adapted the Lec3.2.8.1 cells to grow in serum-free suspension culture, in Freestyle medium. It took some time to get rid of the serum, but now they are growing fine. We now use them regularly for transient transfections for protein production. I don't know what your interest is, but if you want a protein expressed in these cells we could help you with this. We are a core facility working with Mammalian Protein Expression in Gothenburg, Sweden. The adaptation protocol is in a manuscript for publication, but not out yet.

We used glass beads to grow Lec1 in a bioreactor which can hold 10 L with stirring, for the production of alpha-N-acetylglucosaminidase (Protein Expression and Purification 19, 202–211, 2000).

It is fairly simple to turn CHO/CHO Lec cells (and Hek293) into suspension culture. We use chemically defined serum free medium (e.g.CHO Pro293S-CDM or ProCHO 4 Protein-free Medium from Lonza). Adherent grown cells will be harvested by trypsin procedure. Then cells will be seeded in a mixture of regular medium / chemical defined medium (50:50) and cultured for 3 days. Next cells will be harvested and reseeded in a mixture of regular medium / chemical defined medium (25:75) and cultured for 3 days. Thereafter cells will be harvested and reseeded in a pure chemical defined medium and cultured for 3 days. Now cells should grow in suspension. You may test cell viability etc. to ensure the quality of suspension cells.

Using the same methods as given below, we were unable to adapt CHO Lec-1 cells to suspension. It depends very strongly on the particular strain of CHO you are using. CHO cells have an extensive history of natural mutations that have led to the several strains or lines now in culture in labs worldwide. It is important to know which line your cells were derived from. Some of these will adapt well to suspension and some will die without attachment, even when using a stepdown approach.However it is easy enough to adapt to serum-free if you use a media designed for CHO cells only. Not all CHO lines are poor expressors; some can deliver as high as 1 mg/ml., depending on the protein. But if you need the Lec mutation you will be confined to a single strain., Check to see how long this cell has been grown by your lab and any lab who may have supplied it to you. If it has been several years, you may need to go back to the source cell line, as many mutations have already occured since then. In fact it is a good rule of thumb to keep cells in culture no longer than 3 months. Instead of maintaining between experiments,, go back to a frozen master stock for the next experiment. Since these cells are easy to culture after a thaw, you can have them ready quickly. CHO is probably the most problematic, next to HeLa of course, to keep the strain pure (i.e. true to original).

Although CHO is not as easy to transfect as HEK lines, Life Technologies has a new transfecting reagent, Lipofectamine 3000 that was developed to transfect difficult lines such as CHO. CHO cells will continue to express for 20 to 30 days after transfection with a change of media by half every day. In this case you will need a secreting protein ( a good secretion tag if your protein does not naturally express). Collect the media supernate and process for your protein.

CHO cells are the preferred host for production of antibodies because they express well and can be grown serum-free.

There is a mention of growing on glass beads, There are a number of surfaces they will grow on to mimic a suspension: small pieces of nylon fabric, microporous beads of gelatin or Sephadex. These carriers increase the surface area available for the cells to attach.If you need more details I can provide these