cell starvation - which medium is good? (Jul/09/2007 )

Hi

I already put this question on our forum before and at the same time I tried and tried. Finally not succesful. If somebody has experience on that. Actually I use NIH cells for studying the effect of OGP (osteogenic growth peptide). I found the ready protocol from published paper, they use BSA as protein source to replace serum for cell culture when adding the OGP factor, the reason is simple, just for avoid the masking effect of growth factor in the serum. But after spending lots of time on that, I could not arrive in culture my cells in 4%BSA supplemented medium well; cells suffered a lot in that and died in half, so it is immposible to say the proliferation improvement of OGP factor if cells even could not live correctly.

So I would like to ask if anybody have experience culture NIH cells in serum free condition or very low serum condition? COULD you give some suggestion on the correct culture medium for maintaining cell growth?

I attached one photo that NIH cells even could not sustained well after 1.5h starvation duration. Cell adhesion seems really poor.

thanks a lot

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Adding BSA didn't help my early attempts at serum-free conditions, with cells detaching and/or dying. You could also try to step down your FCS over several days (eg seed in 10%, wait to adhere then change media to 5%,2.5%,1%0.1% over four consecutive days. I posted my eventual solution to this problem previously. Good Luck!-JAH

<http://www.protocol-online.org/forums/index.php?showtopic=26286&st=0&p=94047&#entry94047>, "Serum free culture is difficult, and it definitely changes the behavior of most cells. You could try adding ITS+ sol'n in lieu of FBS, adding other GF's as necessary. It's got "...human recombinant insulin, human transferrin (12.5 mg each), selenous acid (12.5 µg), BSA (2.5 g), and linoleic acid (10.7 mg)" We use 1% in DMEM for serum free cultures. Good Luck!
More info? see this link <http://www.bdbiosciences.com/discovery_labware/products/display_product.php?keyID=172> "

Good Luck!-JAH

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Thank you very much JAH. I miss that previous discussion when I search in the forum. Thank you for reminding, I already read that discussion, very helpful, I will do the gradient serum concentration test on my cells as you said. But I have one more question.

My cells could not survive well in 2h starvation period, this starvation period I let them in normal medium without adding serum. Most weird thing this happens from time to time, in some manip they survive well, but next time when I repeat the manip, cells already looks like above photo as I posted. So ----

do you think the seeding density play some role in such capability of surviving the starvation? Everybody say when subconflueny, adding the factor, but subconfluency means how much percentage of confluency? 60%,80% or...

then do you think during the starvation I already should use the low serum medium; then after stavation change to low serum medium supplemented with my factor§

wait for your further help

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After seeding, do you allow your cells to adhere >8 hours before your experiment? This may help cell survival.



Seeding density may have something to do with it. Perhaps you should a preliminary experiment trying different cell numbers per well, and checking the level of confluency after switching to serum-free. Sub-confluency means different things to different people, and these are very crude interpretations of cell density per unit surface area. For my cells, primary chondrocytes, I consider three stages of confluency as approximately indicative of their proliferative activity low confluency(<50%)= log phase, sub-confluent (50-80%), proliferation slows considerably due to contact inhibition and confluent (>80%), which in my cells are completely quiescent, but will become irreversibly senescent and enter terminal hypertrophic differentiation if I switch to Serum free with ITS after 2 days post-confluency. I guess what I'm getting at in general here is that you have to know your cells/system well, and how they behave under various conditions, which requires a lot of unexciting preliminary experiments before you get onto the 'hypothesis-driven' experiments. My notebook probably consists of 80% preliminary experiments, 12% failed 'hypothesis driven' experiments and 8% successes. I shudder to think what my success rate would be if I hadn't spent a lot of effort on the pre-lims, either a lot more flops, or (perhaps worse) false conclusions rooted in not knowing the behavior of your cells in due to control conditions.



While I'm not familiar with the cell you use, some cells produce 'factors' that condition their media. Perhaps in the process of step-reducing your serum, you could do half media changes, by adding an equal volume of serum free media to the culture dish e.g. for a 6-well plate, seed in 1ml 10% FCS, then day add 1ml serum free (FCS=5%), then day aspirate 1ml conditioned media, and add 1ml fresh, serum free, etc. down to negligible FCS concentrations. I would recommend stepping down serum concentration first, adapting the cells to low/serum-free conditions before starting your experimental treatment. Keep in mind that proliferation of most cells will decrease in some relationship to %FCS, so it may be worth doing a series of preliminary experiments so you know what to expect when you test your hypothesis.

I guess what I'm getting at in general here is that you have to know your cells/system well, and how they behave under various conditions, which requires a lot of unexciting preliminary experiments before you get onto the 'hypothesis-driven' experiments. My notebook probably consists of 80% preliminary experiments, 12% failed 'hypothesis driven' experiments and 8% successes. I shudder to think what my success rate would be if I hadn't spent a lot of effort on the pre-lims, either a lot more flops, or (perhaps worse) false conclusions rooted in not knowing the behavior of your cells in due to control conditions. Good Luck!-JAH

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thanks a lot JPH for spending time type all recommendation and suggestions, I will think on that plan my prelim test. Thanks again.
And hope continue to get your idea when I met further problems

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