Dear all,

Just a quick question, which may have been discussed ad nauseam at previous gatherings, but as I have only taken part in a couple, the information hasn't fully rooted in my neurons. We are currently using SF-900 II and III (for Imre's Sf-21's which seem to prefer this medium) from Invitrogen, but our finance department has kindly pointed out that there are better deals on the Ex-Cell 420 medium from Sigma (£18.45 vs £23.60 per litre) to be had.

Obviously, we are a bit reluctant to start playing with a central parameter in our work, but in case the Ex-Cell 420 medium does a similar or better job at maintaining the cells, I'm willing to give it a go, but thought I would like to take vast advantage of the experience of this forums members, so in short - what do YOU use ?

Cheers,

Svend

Dear Svend,

we have been using SF900III since ~1.5 years (before it was SF900II) but we are testing HyClone SFX medium at the moment (Imre's group switched to that one about 1 year ago). For large scale expressions HyClone seems to be equivalent to SF900III (3 viruses tested, expression levels: 1 a bit better, 1 a bit worse, 1 unchanged). The cells seem to have a problem with freezing and thawing in this medium though. We have not yet understood why, since they adapt quite quickly, grow nicely and show slightly better transfection efficiency with a transient GFP expression vector (with FuGene HD) with higher intensities of fluorescent cells (according to our FACS results).

The HyClone medium is clearly cheaper (in the range of 40Euro/L or less, depending on the institute/country discount) but we had long delivery times with our last one or two orders.

We also got some samples from Expression Systems (Tsafi recommended them) which we haven't tested yet. We are also going to test the ExCell medium (Sabine is using the powder) as soon as we can.

As you can see we are also looking for the best solution and it's not a trivial task to change such a critical component overnight.

I hope we can find the best solution based on real data asap. Therefore I would like to ask everyone to comment on my post about the benchmarking of expression systems. I'm not married to any of the components and I would change quickly if there is a better system/component.

Best regards

Hüseyin

Hi Hüseyin,

Wonder if you would share data on cell growth/viability and expression yields between SF900II and SF900III. SF900III is much more expensive for us. When we tested it long ago, there was delivery and QC issue with this media so we continue to use SF900II. However, I heard the current SF900III is better now?

Best regards,

Linda

Hi Linda,

we haven't seen any significant differences between these two media in terms of growth or protein expression levels. With the first btaches of the SF900III we couldn't grow our SF21 cells for more than 4-5 weeks while before we had to thaw new ones only after minimum 3 months. I don't know why they behaved like that.

It's a long story but we had some issues with QC of the SF900II due to contamination in sealed (!) bottles, changes of component supplier, production sites and company re-structuring.

The first batches of SF900III were not as good as promised, but it seems that they have solved some of the issues after some discussions. Maybe this is interesting: during the discussions they admitted that there is no QC after production of each batch where they would test the protein expression performance. The just test the growth kinetics and cell densities (which of course is not telling you anything how good a virus would infect and how much protein you could get). So you need to be aware of this, that's why we test each new batch with a difficult protein before ordering a larger volume. They promised to change this in the future but I don't know if they are testing now.

Regarding the price, as usual most companies have different policies and discounts depending on the countries and institutions. With some companies It's not possible to get the same discount e.g. for two EMBL sites in Germany and France.

Best regards,

Hüseyin

Hi Hüseyin,

Thanks for the information.

Best regards,

Linda

Dear Svend,

we have been using ExCell420 from 2006-2010 without any problem ever. We switched to SF900 II for price reasons.

In june 20111 problems started, 10L empty productions, reduced growth etc.

We than used SF900 II liquid (list price) Expression went back to normal but still we were not able to thaw SF9 cells, and High Five cells still in culture had waves in viability between 85 % and 92% and a constantly increased cell size over weeks. When we thawed fresh H5 cell size was already increased after 24h !

We then tested several media, decided again for ExCell420 (SF9) and ExCell405 (High Five). We compared expression levels of 5 proteins, no difference.

Every cell line was thawed freshly in this medium, and immediately showed constant viability > 95% and cell size reduced to normal

We are currently running 2 x 5  L prodcutions / week, the cells are perfectly healthy and absolutely stable.

Dear all,

I have found this discussion very informative as I had recent problems for growing my insect cells and I thought I was doing something wrong!

While I am not doing a lot of insect work, I have followed the inducible system (no virus) that a postdoc nearby established.  Briefly, It consists in transfecting S2 cells (co-transfection of pMT/BIP/V5-His from Invitrogen and pcoHygro for selection) into Schneider's medium, to carry on in selecting the cells with Hygro in the same medium prior to adapt the cells in Express Five SFM. Once the cells have sufficiently expanded, the induction is induced with copper and the protein is purified from the supernatant. While the combination of cells/media might not seem ideal, the adaptation of the cells is just a matter of days and worked very well in my friend's hands.

The system worked quite well for my first attempts with a protein (before June 2011) so I decided to try another protein and the same protein with a different tag (after September 2011). I started to notice a problem in the proliferation of the cells and the level of expression was dramatically reduced. I intended to do some transient transfections to exclude problems with the selection but because I had too much work, I just put the insect system on hold.

The discussion on the media from Life Technology gives me hope. I am going to look at other media and give it a try when I have more time.

Many thanks to all!

Patrick