|we are trying to adapt CHO dhfr-( ATCC CRL-9096) cells to SFM, suspension condition sequentially and have done it successfully till 1% FBS with the population doubling time(PDT=20hrs in static flask)remaining almost the same as the original unadapted cells.Weaning from 1% to lesser concentration seem to increase the PDT to 80 hours(in uncontrolled bottle culture). can anyone suggest why this is happening and is there a different way to adapt these cells?|
we are using the customized SFM for CHO cell suspension culture, giving medium change every 3 days.
|That cell line has an insulin requirement and it is somewhat picky about its iron source. If you add one of the commercially available ITS supplements you should get out the final amount of serum.|
|Thanx a lot Trish for the help, but this is for your information that we are supplementing the SFM with glutamine, and ITS too. Do you think the cells need something more for their growth or is it that we should manipulate the concentrations of these ingredients itself??|
And sir can you give me your personal e-mail id for further contacts??
|Your message is very similar to one posted by Shikha on 5/17. The major difference in that posting and yours is that Shikha specifies that the 80 hour doubling time is seen in a shake flask culture.....if your are using a shake flask culture you probably have the same problem. When serum is in a media it provides some viscosity to the media which, in turn, provides some cushioning for the cells as they are shaken. When that is removed the cells are torn to pieces by sheer forces....this is true for spinner flasks at high RPM and in bioreactors as well. To replace the viscosity you need to add a sheer protectant. The most commonly used sheer protectant is Pluronic F68. It is sold as a sterile 4% solution by most of the media supply houses. You will need to add 10-20 mls/L to replace the sheer protection provided by 10% FBS.|
My email address is firstname.lastname@example.org and it may take a couple of days but I answer all of my email...provided it gets past my virus protection software.
I think you are Carrying the process in a simple bottle
in uncontrolled way, Not exactly knowing the RPM and
other things. some strains of cho require high amount
of oxygen also for their doubling time. I doubt you
are supplying oxygen. The actual problem starts in all these cases only when you wean serum completely. In
My present lab From last month 25th Till date we have
successfully completed the changing of adherent culture to suspension without any serum . We have shifted from 10% to 1% serum directly and then total
serum free conditions. I completely agree with what
trish says. For that one should learn media optimisation and you should select a few components
which your cells require May be amino acids , vitamins and trace elements. Then with pluroniucf68, Bit of tween 20 ETC. untill and unless unknow this optimisation studies completely , both physical and physiological parameters you cannot be successful , which needs lot of time and patience. Better in your case in DR. Reddys you can use baffled flasks to start
your experiment with if you do not have small spinner flasks. As trish mentioned for there were more than 50
ingradients to feedle and you should Know exactly when
to use which ingradient,.
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