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Position: Discussion forums > Bioscience biotechniques discussion forums > Cell biology and histology methods forum
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Anti-clumping agents for CHO-K1 suspension cells
Posted by: Anna Albert (IP Hidden, New member, 3)
Date: April 25, 2005 06:58AM
Hi, I am establishing a CHO-K1 suspension cell line, but am finding that the cells are forming large clumps which are difficult to break up. Does anyone know of any methods/anti-clumping agents which could help to prevent this from happening?
Re: Anti-clumping agents for CHO-K1 suspension cells
Posted by: 5'-ATCG (IP Hidden, Unregistered user, )
Date: April 26, 2005 05:08PM
Try some reducing agents like 2-mercaptoethanol which has been extensively used in lymphocyte culture (easy to form clumps..).
Re: Anti-clumping agents for CHO-K1 suspension cells
Posted by: aphio-igor (IP Hidden, New member, 6)
Date: March 16, 2008 04:52PM
I agree with the BME (beta- mercaptoethanol) comment, noting that required concentrations are minuscule; our lab currently uses 52uM BME as a 1000x stock, and Invitrogen/ Gibco sells a 55uM BME as 1000X stock (Cat# 21985-023) -- I have no affiliation with their products/ am not a salesperson for them, so standard disclaimers apply, etc etc etc.
There's three other products that I've used with GREAT success. From least effective to most, I've used another Gibco product, "TrypLE Select" (an animal-free, 'wanna- be trypsin' -like disaggregation solution) actually IN the culture medium. Serum won't bind it up, but you'd need somewhere in the neighborhood of 10 to 20% by volume. While it won't lyse the cells per se, it does do a great job of chelating extracellular Ca and Mg ions... which in turn MAY decrease your PDR (population doubling rate). I actually used this with decent (not great, but hey it worked) success on Cho-K1's in CD-CHO media (with HT supplement for the nucleotide production/ growth rate) with TrypLE Select at 10% by volume, noting it was shaking at 110rpm and was seeded at 4e5 c/mL as a minimum to 2e6 c/mL as a maximum. The later two products are intended to be used as a pair: Accutase and Accumax, both by ICT (I think it stands for Innovative Cell Technologies - the Accutase/max is right, so google if I'm wrong here). The Accutase is a Cell Culturist's dream - the company apparently knows it and charges accordingly... Regardless, while it doesn't need to be serum- neutralized, like the TrypLE Select above, I suggest centrifuging your suspension, adding ~10mL of the accutase to the cell pack (pellet, minus supernatant), then qs to your desired volume with fresh media. Why? Merely the cost of the stuff, not that it can't be added to the culture itself (haven't tried it, so can't say either way on that), but rather that it uses less to "spin and soak" as written. The Accumax is intended to be used more for in-culture use. That is, it is intended to be added to culture medium much like the TrypLE Select above. This stuff is likely just what the doc ordered for your application - I prefer it hands down for flow cytometry and bioreactor Mc|ab production efficiency. Likely too geeky of a comment here, but it is the only product i've found that results in a single- cell suspension without altering the collagen, integrin, or cadherin profiles of the cells when compared to their progenitor adherent line. I hope that helps you out more than it confuzzles. Kind regards
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