why my BSA contained medium cannot nourish my cells - (Feb/02/2007 )

hello everybody. I am new comer but I like here.

recently I met the problem with my new test, I repeat several times and the problem remain the same. The story is like following:

As I am investigating a new peptide synthesized ourself, which is surposed to improve the proliferation of my cells. So I follow the classic researching protocol for investigating the effect of growth factor, I should not use the serum contained medium. So I decided use the Bovine serum albumin (BSA) as substitute of serum to nurish my cells during 48 hours culture, which acturally also followed some protocol in published paper. But I met the problem after I add the BSA into the cell culture, the cells shrinked and in some location cell even died. I am really anxious by this problem, for I really try to imagine all possible reason and take care all and finally the problem still remains. I don't know if somebody here who had similar researching experience could help me find the knot . My protocol is:
1. osteoblastic MC3T3 E1 grow in minimal essential medium (MEM) supplemented with 10% fetal calf serum (FCS) were seeded in 16 mm multiwell dishes at 2 x104 cells per well and incubated in the same medium at 37 °C in CO2/air.
2. For the initial 46 h, the cells were incubated in the same FCS-containing medium. The cultures were then washed and followed by replacement of the serum-free MEM with a medium containing 1% fatty acid free bovine serum albumin (BSA) (Sigma Chemical Co., St. Louis, MO; catalog no. A-7030) with or without tested peptide.

thanks a lot for your time and attention

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Hi lilylille,
I am not familiar with the specific type of cell you use, but perhaps you could try a serum-free media: I have used "Ultraculture" from Cambrex in the past, for a tricky line of cells, and they really grew well!
http://www.cambrex.com/Content/Documents/B...ltraCULTURE.pdf
Good luck!!
mito 1

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thanks a ton for such rapid answer.

I want to confirm if you mean that if I use the serum free medium which you proposed and then I don't need add BSA any more for nurshing the cells? So by this way I can avoid the problems which brougt by BSA?

thanks again

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The Ultraculture has all the bits and pieces that go into into serum (ie: defined mixtures of bovine proteins), so you should not have to add FCS or BSA. Like any other type of cell culture though, it ALL depends on the cells You do have to add l-glutamine. There are probably other types of serum-free media out there, and I'm sure that others on this board have used them: this is the media that I have used successfully. Double-check the pdf file, to make sure that there are no other elements that can interfere with your experiment.
Cheers!
mito 1

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Thanks again Mito1.

So normal culture medium without adding serum is definitely different from socalled 'serum free medium' product??? Actually I just want to know if I am obligatory to buy this 'serum free medium' product, or the normal culture medium without adding serum but adding BSA could work too. Could you anwser this. thanks in advance

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Good morning lilylille,

Media without serum is not the same as "serum-free medium": the former is just base media (eg: DMEM), without any serum added. The latter (SFM for short), is specially-formulated media that has defined components in it: that is, instead of adding serum (which can vary from lot to lot, and has many less-regulated proteins, hormones, etc), the precise components are added in measured amounts by the manufacturer. These components usually include BSA, as well as other proteins, lipids, insulin, transferrin, etc. BSA alone is protein, but may not provide all the other "yummies" that your cells need.
Also note that there are "reduced-serum media" formulations on the market, which require serum but in lesser amounts.
If you are following a published procedure, then be sure that the BSA you are using is cell-culture compatible.
I can't give a definitive answer of yes or no, to your question of adding BSA to normal media, because I don't know enough about the cells or conditions involved in your work. The nice thing about SFM is that you don't have to worry about lot-to-lot serum variations, so it often works out cheaper!
If you decide to switch from a serum-containing media to a SFM, you need to wean your cells off of the old formulation (don't make them go "cold-turkey").
Again, double-check the published protocol to be sure you are not missing anything.
Best of luck - don't hesitate to ask more questions!

Cheers (and off to get more coffee!)
mito 1

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thanks a lot for such elaborate explaination about the SFM, I think I know better about it now. And following your advice, I rechecked the protocol performed in pulished literature, their description is like following
1. seeding the cells in multiwell plate and cells were incubated in 10% FCS containing a-MEM in initiql 48 hours;
2. then the culture has been washed and kept for additional 2 hours under serum free condition
3. this is followed by replacement of serum free a-MEM with a medium containing 1%BSA and with certain tested peptide factors.

So from above description, now I would think they used really serum free a-MEM , but not a-MEM without adding serum. Do you think so? And they starve cells only 2 hours from 10% FCS to 0% FCS, such way is safe or not? The cells I used is MC3T3 E1 osteoblasts cell line. If they really use the serum free a-MEM , so it seem I should search it and your proposed ultraculture will not so pertinent to my case?? I really feel lost with cells, I still don't know them.

thanks for your time and patience; I am really happy to find this place .

P.S. just now I searched the serum free alpha-MEM ; but acturally it seems no such commercial product existing. So that seems their medium is just alpha-MEM without serum?? Could you help me make a guess about their protocol??

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Serum is the product of clotted blood. Blood clots form in response to a cutaneous wound. The growth factors derived from platelet degranulation (primarily PDGF, but also TGF-beta, IGF, FGF, EGF, etc.) are what make your tissue culture cells grow. These factors also drive the early stages of wound healing. Fetal wounds heal more quickly than adult ones, and without scarring. One of the reasons for this is hypothesized to be that the cells activated by fetal serum grow more quickly than those of adults, due to a different balance of cytokines. Thus cells grow more quickly in FCS than in NBCS where they tend to grow bigger and slower.
FCS is the only one of the three that does not contain antibodies.
ECM factors, such as fibronectin, are also present in serum.
-Heat treatment inactivates complement and should always be done with FCS!

These cytokines are not present in BSA.

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I really hate to guess about protocols that I haven't seen , but it sounds as though when the authors say "serum-free conditions", they mean a-MEM without serum. So this would be your regular a-MEM that you using, but without any added FCS. It sounds like they want the cells to undergo a short serum starvation period: that is, a length of time in which the cells are not exposed to any "serum goodies". Then, certain specific reagents (BSA, peptides) are added back on. If the authors say "2 hours", then the cells should be fine. Again, I emphasize that this is only based on the info you provided. It MAY be that they are trying to get the cells to the same point in the cell cycle, but I am guessing
Good Luck!
mito 1

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thanks tfitzwater, but I didnt really got what you want to say. anyway I already totally lost.

thanks mito1, sorry for forcing you to guess. I know such require is cruel. By my different test with different controls, I more and more sure that protocol in published paper, I could not repeat, I could not say there are something wrong in their protocol, but at least my cell dosnt like that, even we use the exactly same cell line as them. I repeat that 2 hour starvation step sveral times, after that 2 hours, cells all already partly died. So from 10%FCS to 1%BSA by just 2 hour starvation; at least my cells dont like and cannot adapt so quick!

So I will order the serumfree media and systematicly construct my own protocol now.

thanks

I am in france; always have to wait one day to get your advice.

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