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Position: Discussion forums > Bioscience biotechniques discussion forums > Cell biology and histology methods forum
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CHO-S cell
Posted by: annkitti22 (IP Hidden, New member, 1)
Date: December 26, 2007 09:40PM
Dear Sir/Madam,
I have an important question about CHO-S cell (cat No.11619-012 Gibco) derived from Invitrogen. We tried to grow them in monolayer but failed. The media we used was F12 Nutrient mixtures (Ham) , cat No 11765-054 Gibco) + . Do you have any suggestion? We also used DMEM (4500mg/L D-glucose, L-glutamine and 110 mg/L sodium pyruvate , cat No 11995-065 Gibco) + 10% FBS which make the cells adhere to the plate but they grow very poorly. Your kind suggestion of the appropriate media or adaptation of the above media will be greatly appreciated. We look forward to hearing from you as soon as your convenience. Regards, annkitti22
Re: CHO-S cell
Posted by: aphio-igor (IP Hidden, New member, 6)
Date: March 16, 2008 05:25PM
This may be a silly question, but is it a typo that you used F12(Ham's) without serum? If there's no serum, chances are that's the issue. Also note that these cells were selected for their serum- free adaptation/ modification (SFM). Cho-S is a suspension-preferring subclone of Cho-K1; while it can easily revert back to an adherent morphology, you are essentially re-adapting the cells from a suspension growth back to an adherent one. With any medium adaptation, one can expect a few passages with poorly defined morphology, if not biphastic growth properties.
I apologize if that was worded poorly - it was not meant as such. What I'm simply trying to say is that the conversion back to an adherent state may require exceptionally large seeding densities into the appropriately- sized T-flasks. Just as a suggestion, as a "surely they'll stick with this!", try using a 20% serum medium for a passage, then revert to 10% from there. Additionally, CHO cells have been reported as having poor metabolisms - while I doubt it will do anything for adhesion, it may make the cells 'happier' to supplement with additional L-glutamine or even Sodium Pyruvate. Again, those two components are merely for 'happier' cells, but note that the L-glut degrades very quickly, especially in light. Just some thoughts - I'm interested to hear what others say, also. Kind Regards
Re: CHO-S cell
Posted by: VPIlabs (IP Hidden, New member, 2)
Date: April 2, 2008 07:12AM
Hi,
I have a problem concerning CHO-S cells. I have been trying to transfect adherent CHO-K1 cells with multiple transfection agents, which worked. After that I'd like to dissociate these transfected cells to study internalisation of the transfected receptor (encoded on the plasmid) in suspension, but I have no surviving cells after this trypsinisation. I usually trypsinize them 4 hours after transfection and grow them in fresh medium till 24h post transfection. Is there anyone who has some suggestions or who has already done this? I am able to keep non-transfected cells alive, so I think the combination of trypsinization and transfection just kills my cells. Another option is to buy CHO-S cells wich are in suspension, but I have no stirrer inside our incubators. Is it possible to grow these cells without stirring them? Or will they start to adhere? Also, do you need the serum-free medium or can you use F12 or other (cheaper) media? Thanks in advance for your reactions!
Re: CHO-S cell
Posted by: aphio-igor (IP Hidden, New member, 6)
Date: April 2, 2008 12:55PM
Hi, VPIlabs-
This may be a silly question, but when transfecting, which transfectant agent (chemical, kit, product name, etc) do you use? The reason I ask is that some products function via a 'superglue- like' method (i.e. Polybrene, DEAE-Dextran derivitives, etc - mostly intended for viral vectors), other products work through membrane binding and engulfment, be it via lipid raft formation, receptor binding, or a myriad of other methods, and yet others function through membrane disruption and so- called "weakening". Enough background rambling - the trypsin you use may be "punching holes" in the membrane and lysing the cells you want. Why does the trypsin only affect the transfectants and not the residual parental, untransfected cells? The transfecting agent/ product may be (emphasis on MAY be) only affecting those cells that get the plasmid - kind of makes sense, as the non- transfectants didn't get transfected for the same reason that the others did, that is, through action of the agent. Bottom line is, the transfectants may have "weakened" membranes already, to get the plasmid punched through. The trypsin, by chomping up the extracellular proteins, may induce a ton of "bad ju- ju" (technical jargon, I know...), including but not limited to apoptosis, etc. How to solve it: First, you can spin out the trypsin from the trypsinized cells, then resuspend the cell pack/ pellet in fresh media. Remember, trypsin isn't used up during the debridement process, so it will keep chewing those cells' proteins until something else stops it. Serum has anti-trypsins, as well as tons of BSA (bovine serum albumin) which essentially dilutes out the cells' proteins from the total pool of proteins available for the trypsin to chew on. As you're using Serum- Free media, I'm guessing the trypsin (while diluted out via fresh media) may not be fully inactivated when you initiate your suspension culture. Oh, and with the trypsin/ transfection being so close together, I'd limit the spin to 125 x g. You can spin for 10 minutes if needed, but don't go nuts and squish them at 200 x g... sorry if rude, but I'm speaking from experience when I say "If you find you've done all this work, and then get the cells squished, that really stinks" Second, you can wait more than 4 hours post- transfection before debridement/ trypsinization. I doubt that's the issue, and depending on what receptor you're looking at, it may be counterproductive to wait more than 4 hours - I don't know. What I can say is that the interim period between transfection and trypsinization will allow the cells to start producing proteins again, aiding in membrane stability. Third, you can use a different (synthetic?) debridement agent. Trypsin is great, don't get me wrong, and relatively cheap besides, but *as a last resort*, you can try something else - I'm a huge supporter of TrypLE Select, a Gibco/ Invitrogen product (no affiliation besides being a VERY happy user). The TrypLE Select dilutes out with the addition of fluids (media). There are a whole myriad of other products out there, and I can point you in their direction if requested. As for the CHO-S option, you can use shake- flasks at ~110 to 140 rpm in place of stirrers. Note that CHO cells (any type that I have dealt with, and - for better or worse - that's more than I'd care to admit) are known for being "stagnant buggers"... that is, you may need to spike them with HT supplement to get them kick- started into DNA replication again. The... oh goodness... H = hypoxanthine? T= thymidine? just guesses - I've got a 3-day-old son, so forgive my memory lapse... anyhow, that HT sup will provide the needed 'juice' for the DNA replication. Also, CHO's tend to be slow in producing L-glutamine, in general. It isn't like they're going to up and die on you, but a few mL's of L-glut are worth their weight in gold when trying to get "happier" cells. So what I'm getting at is that the Cho-S cells would *likely* work, but the CHO-K1's are likely better for the transfection itself, and the subsequent morphologic change/ media- change is much less of a problem - lesser of two evils, if you will. Personal opinion, and I'm sure that others will disagree wholeheartedly. As for growing CHO's in suspension without stirring, again, one can shake them with shake- flasks. I suppose you could use T- flasks, and they should work just fine, but be prepared for clumpy cells the first several passages. (it gets better as they adapt to the media) As for cheaper media, (i.e. Ham's F12, F12-K, etc.), I really wouldn't risk it. As with all of this above 'book' that I've written, it is just one guy's experiences and opinions... but saving a buck now isn't worth the zillion bucks later for having to repeat the whole darned thing again. Just my opinion, sorry if it came out poorly. To help with costs, my experience is that CD-CHO +HT is your best bet. I don't know what receptor you're looking at, so it's hard to recommend media - you may get oddball results with some of the media's components - but *generally* speaking, the CD-CHO with HT is your best bet. A close second is Mach1, but that's intended more as an antibody- producing medium. Other than that, unless I've done it wrong several times over (and trust me, that wouldn't be too far of a stretch, nor would it be the first time!), Ham's F12 won't be enough for your cells in a serum- free environment. They'll be there, and likely some will be alive, but when transfecting and looking to select the clonal population, I'd sooner hedge my bets with a different media than risk all to save a few bucks - sorry again if that was worded poorly, but I'm just trying to save you grief in the long- run. Speaking of grief in the long- run, our newborn has "volunteered" me for diaper duty... oh the joys... Best of luck and kind regards, Pete Gramp (aphio-igor)
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