Hi, all,
There are plenty of bubbles on the surface of cell culture medium in my experiments. I cannot get rid of these bubbles because the whole system was continuously gassed with CO2, meaning the bubbles are produced all the time.
It seems that the bubbles don't have severe bad effects on microscopic imaging of the cells. But I don't know whether these bubbles will affect the cell behaviour, the cell proliferation etc.
Any of you have any comments on this?????? Many thanks in advance!
another question: how to get rid of these bubbles?????? If I could get rid of these bubbles, that would be perfect then. Any one knows anything?????? Thank you :)
Kind regards,
Jing-Huan :)
Dear Jing-Huan,
First of all: how do the bubbles get in the medium?
I guess you have a gass inlet in the medium itself?
A solution would be to very simple not put the gass inlet in the medium but over the surface of the medium.
In CO2 incubators the whole chamber is gasse, hence the flasks in them are gassed.
bubbles are caused by the serum added to the medium. Bubbles distort the microscopic views only little but when you want to make micrographs they will not be nice. Bubbles eventually wil burst and cavitate. This has generally no severe effect on the cells but it introduces motion in your medium. If that is not an issue, I would not care. If you do videomicroscopy I would use another technique.
getting rid of the bubbles?
Try a dry sterile swabstick (a small cotton ball on a stick).
Try a sterile sonic needle but do not dip it in the medium otherwise you sonicate the cells.
Hope this helps
Are these cells cultured in suspension or monolayers? In any case try to passage them as gently as possible and avoid the bubbles at this stage already.
bubbles will cause some effects on cell behavior. cells attached with bubbles will get shear force when it broke. surface area of bubble is important in diffusion of composition like oxygen, CO2, etc.,, from bubble to liquid to get equilibrium.
Thank you Stefan for detailed explanation and suggestion. I study cells underflow, and take video, so the bubbles will affect the video then even though the burst of bubble is fine for the cells according to your opinion.
The bubbles were continusly introduced into the medium because I pass the medium through a gas module and after the gas module, the medium have bubbles on surface, I have to add CO2 gas to the medium for cells because the cells are not inside incubator. I am thinking to connect a filter to the gas module to block the bubbles entering the tube, but allowing only medium flow.
Thank you again. Your information is useful to me anyway. I think if in the end I cannot get rid of the bubbles, I can live with them then without big worries of cell behavior anymore :)
One problem is that (as Stefan said), the bubbles are caused by the proteins in the serum having a surfactant effect, like detergent. I have heard that too much frothing can denature some of the proteins, which are also the growth factors for your cells, so there might be some bad effect.
It sounds as if you are aiming to have a steady-state cell culture system. One way this has been done before now was to have a very slow, steady flow of medium plus also a very slow steady flow of CO2-air mixture over the top (not going directly into the liquid, as Stefan also said).
We used to do time lapse video microscopy which is quite similar to your experiments. In our system we had a heated stage microscope. The cells were normally cultured in 6 well dishes or petri dishes. The whole microscope stage was placed under a gassed custom made plexi hood. If you like I can go to the lab and send some pictures. Conceptually it is rather simple and cheap to make if you do not need to do a lot of experiments. A transparent PE box will do the whole trick. Find a box which can fit on you microscope stage. Make sure the bottom is crystal clear and still enables focussing on the cells. Open the lid. Place the cell culture in there. Close and seal the lid with breathable tape. It is important to have the box gassed which can be easily done with a syringe needle which can be heated and pushed through the plastic. Otherwise drill a whole, insert a nipple to connect the gassing hose. Your all set for a bubble free experience.
Filtering the medium is indeed an interesting suggestion. Realize however that your filter should be big enough since particles in the medium and detached cells might clog the filter (eg Millex-HP Filter).
hope this helps
My experience is that cells dont like bubbles. Bubbles will make a "dent" in the medium surface causing cells to refrain from and not grow in that area. Probably has something to do with surface tension on top of the cells. I would expect that these problems depend on the volume of medium that is used on the cells. I would try to prevent bubbles in your system as it is already artificial enough to culture cells ex vivo.
Cheers, Toine.
Thank you Stefan for detailed and clear explanation of your gassing system, useful information :). I guess I can gas the medium surface in a bottle, then connect the bottle to the wells (the wells are also inter-connected, I am using Kirkstall's Quasi-vivo900 6-well plate). Then the medium flowing through all the inter-connected wells should be controlled for PH value.
Yes, I agree with you for filter removing of bubbles, there are risk of clogging of the filter. This wouldn't be a good solution.
In my experience, bubbles are not as problematic for monolayer cultures as for suspension cultures.
To get rid of the bubbles, try sparging through a CO2 permeable silicone membrane.
The book "Cell and Tissue Engineering" written by D.Eibl is a valuable source to tackle this problem, too.
Hello Jing-Huan Wang,
you could try this: http://www.permselect.com/products
Generally, a hydrophobic hollow fiber module should work.
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