How to grow adherent cells as a suspension???

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MadScientist74
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How to grow adherent cells as a suspension???

Hiya!

Need help urgent...

I am looking for a way to grow U2OS cells (adherent) in a manner where they do not attach to the substrate base.

What flask materials or treatment can I use?

I have tried using the normal petri dishes for growing bacteria but the cells attached to grow! Short of using Falcon tubes, I am running out of ideas already!

Appreciate your inputs...

Cheers!

Galicola
Galicola's picture
I know that adherent cells (I

I know that adherent cells (I use CHO) detach when grown in serum-free media (SFM).
You can buy different kinds from Sigma, JRH, Biowhittaker, HyClone, Gibco and probably others.
Ask them which SFM suits your cells, but the only way you can know for sure is just try several kinds and see which one is the best one for you (in terms of viability, growth, aggregates etc...).

Good luck

genxpress
genxpress's picture
Hi

Hi
I am too trying to adapt a cell line to suspension. It is growing well in suspension, the problem is, huge clumps of cells are forming. Can anyone tell me how to get rid of this clumping. Because, my further experiments are based on this cell line.

genxpress
genxpress's picture
Hi,

Hi,
Use of serum free medium may change the characteristics of a particular cell line. It also depends on the experiments you wish to do. You can grow them in spinners which are siliconized. Iam growing a adherent mammalian cell line in siliconized spinners. They are growing well.

MadScientist74
MadScientist74's picture
I am trying to 'grow'

I am trying to 'grow' adherent cells in suspension as I am looking into attachment independent growth properties of the cells.

That's why I cannot 'play' with the media components.

I dun mind clumps.... and I do expect clumps as in nature, any pressure would result in a reactive outlet elsewhere... so, clumping is a 'reaction' to the cells being unable to attach to the base substrate.

Anyone got any more ideas???

Cheers!

Galicola
Galicola's picture
About cell clumps - I know

About cell clumps - I know there's an anti-clumping agent (that's its name) from Invitrogen.

I haven't used it, so I don't know how well it works.

Richard Taylor
Richard Taylor's picture
You could use a rotary cell

You could use a rotary cell culture system RCCS.

samm
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You could try silanized

You could try silanized culture ware - essentially a sort of "teflon coating"! I do not know how your cells will take to it, but as a trial, you can use borosilicate glass petriplates, coat with Rain-X (the same stuff applied on car windshields), dry, and culture cells. Am not sure if it works on plastic/PE/PP surfaces, but again you can try. Rain-X is the brand name in the USS, but I'm sure there will be similar products available.
Also, even if you cannot switch to SFM, try reducing FBS amounts - often you can go down to half or one-fourth original levels, but do it gently, over three or four medium changes.
Finally, keep us posted on what happens - this is rather interesting.

MadScientist74
MadScientist74's picture
samm wrote:You could try

samm wrote:

You could try silanized culture ware - essentially a sort of "teflon coating"! I do not know how your cells will take to it, but as a trial, you can use borosilicate glass petriplates, coat with Rain-X (the same stuff applied on car windshields), dry, and culture cells. Am not sure if it works on plastic/PE/PP surfaces, but again you can try. Rain-X is the brand name in the USS, but I'm sure there will be similar products available.
Also, even if you cannot switch to SFM, try reducing FBS amounts - often you can go down to half or one-fourth original levels, but do it gently, over three or four medium changes.
Finally, keep us posted on what happens - this is rather interesting.

Thanks!

I do not have a rotary cull culture system here... but I do have the chemical to salinize... just the same, I have no idea if it works on the plastic plates (whether it would 'melt' the plastic) or if the cells can take it (as I think it is toxic).

So, still at ground zero....

Thanks for the suggestions so far... would appreciate if they kept coming!

Cheers!

genxpress
genxpress's picture
hi,

hi,
i too use silane for coating the spinners in which i grow cells. after siliconization of the spinner, i thoroughly rinse it with water and then autoclave it. but, silane is used for not attaching cells to the surface of the spinner but not for resolving the clumps

MadScientist74
MadScientist74's picture
genxpress wrote:hi,

genxpress wrote:

hi,
i too use silane for coating the spinners in which i grow cells. after siliconization of the spinner, i thoroughly rinse it with water and then autoclave it. but, silane is used for not attaching cells to the surface of the spinner but not for resolving the clumps

Ok, thanks! From your desciption, it looks like the silane is non-toxic!

cheers!

Protoplast
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Hello, just a thought, i work

Hello, just a thought, i work on fungal cells, and to stop our cells from clumping together when we grow them, we add a small amount of TWEEN80 (a detergent) it usually works O.K. maybe that is a good idea?

MadScientist74
MadScientist74's picture
Protoplast wrote:Hello, just

Protoplast wrote:

Hello, just a thought, i work on fungal cells, and to stop our cells from clumping together when we grow them, we add a small amount of TWEEN80 (a detergent) it usually works O.K. maybe that is a good idea?

I think that Tween would be a lysis agent for mammalian cells.... yes, I do remember using Tween for fungal cultures in Biochem Engineering.

Thanks for the input... still looking for a feasible solution.

samm
samm's picture
What happened with the

What happened with the silanization?
Yet another suggestion: I've just started using trypsin-free cell dissociation agents - there are a whole bunch of them available now. I guess they are a proprietary mix of chelators and very mild (non-cell permeabilizing) dispersants. Perhaps you could try using them in culture, though i have no clue what might happen. The longest I've done it is ~6 h.
-sam

MadScientist74
MadScientist74's picture
samm wrote:What happened with

samm wrote:

What happened with the silanization?
Yet another suggestion: I've just started using trypsin-free cell dissociation agents - there are a whole bunch of them available now. I guess they are a proprietary mix of chelators and very mild (non-cell permeabilizing) dispersants. Perhaps you could try using them in culture, though i have no clue what might happen. The longest I've done it is ~6 h.
-sam

Salinization wasn't too compelling to start off with... so I left it for the time being.

In fact, I just found a damn expensive plate that is designed to do just what I wanted! But it costs about USD$15 per plate (12 well plate).

My preliminary trial show that the cells do not spread and seem to grow ok... at least up to a week. Have not tried to do other procedures yet.

debien
debien's picture
I'm not sure if you're still

I'm not sure if you're still dealing with this but in the past when I wanted to grow cells under non-adherent conditions, I used "Poly-Hema" on my regular tissue culture dishes or flasks and this coating prevented cells from attaching. Procedure follows:

Poly-HEMA Coating of Tissue Culture Plastic

Poly (2-hydroxyethyl methacrylate) (Sigma #P3932)
Cell adhesion is blocked completely on plastic treated with 100 l/ cm2 ?20 mg/ml poly-HEMA.
Substrate treated with 100 l/ cm2 ?0.02 mg/ml poly-HEMA is nearly as adhesive as TC plastic.

1) Dissolve 1 g Poly-HEMA in 50 ml 95% ethanol overnight at 37?C. This makes a 20 mg/ml solution.

2) Clarify solution by centrifugation (2,000g, 20 min.).

3) Apply desired dilutions of poly-HEMA in 95% ethanol to culture vessels and dry on a level, vibration-free surface. Warm to 37°C if desired. UV-sterilize as necessary. For non-adherent conditions, use Poly-HEMA at 20 mg/ml.

4) Rinse vessel with PBS. Seed cells (sparsely to avoid aggregation) in coated culture vessels.

Good-luck!

jonatmudd
jonatmudd's picture
I found that polyHEMA (Sigma

I found that polyHEMA (Sigma P3932) is *excellent* for preventing cell adhesion and it seems to be compatible with growing cells in suspenion.
polyHEMA, in my hands, has proven MUCH more effective than any silanization protocol I've tried.

My polyHEMA protocol is as follows:
add 40 mg polyHEMA to 2 mL EtOH. mix overnight on rocker. (it takes a long time to dissolve)

To coat a dish, cover with a thin layer of polyHEMA/EtOH mixture. Be sure that evaporation happens very slowly by putting your dish inside of an (almost) air tight container.
If the polyHEMA solution dries to fast it tends to coat very non-uniformly in some sort of strange patterns,which causes optical issues--your cells will be hard to view.

Multiple coats--2 or 3--is superior to 1.

Cheap and easy solution..try it out!

MadScientist74
MadScientist74's picture
Thanks for the replies!

Thanks for the replies!

I've "given up" on this aspect but it is something new that I am learning...

Cheers for the replies!

heehawmcduff
heehawmcduff's picture
The teflon coated pots that

The teflon coated pots that you can get work pretty well for macrophage culture (which are typically pretty adherent over long culture periods)

Greven
Greven's picture
If i make PolyHEMA plates, do

If i make PolyHEMA plates, do I need to use it direclty or can i store it in +4 for a few days until i need  it?

asif123
asif123's picture
Dear

Dear
Its really good idea to use detergent (if it is not harmful for the cells), could you please broad the small amount of tween 80.

Thanks
 

asif123
asif123's picture
Dear Samm,

Dear Samm,
Do you have any idea about the use of 0.2% EDTA solution in the suspension culture till the end of the process.

Thanks
Asif

samm
samm's picture
Hi Asif - you need to specify

Hi Asif - you need to specify amount of EDTA as a molar concentration (usually EDTA is made as a stock in water at 0.5Mat pH8, for cell culture, its diluted in PBS or HBSS). Also, cell type,, how long you want to grow them, and purpose for the adherent-->suspension switch. As you might have appreciated from reading this thread, actively growing - i.e.expanding adherent cells in suspension format is not a trivial undertaking!
Ciao
-sam

asif123
asif123's picture
Dear Samm,

Dear Samm,
Many thanks for response; I am going to adapt the adherent BHK-21 cells to the suspension culture. I have gone through various literatures talking about the adaptation of the same cell line.
I want to take the advantage of the chelating nature of the EDTA to keep the cells in suspension or at least to avoid the huge clumping in the initial stage of adaptation. I have gone through the trypsine free cell disloging agents, they contain the chelating agent  either inPBS or HBSS.
As the ion channel is very important for transport of various cell need and remove the toxic release, I am afraid that use of EDTA can block the cell metabolism.
I will be very grateful, if you will suggest me in this regard.
Asif

samm
samm's picture
The highest EDTA conc I have

The highest EDTA conc I have used has been 100 mM - but that has been to detach and maintain a single cell culture for a short period of time, without affecting surface receptors.
 You can perhaps try the PolyHEMA protocol outlined below - as you rightly said, eliminating Ca usually results in cell stasis at best, cell death at worst (which can also happen when cell-cell contact is lost in suspension growth cultures, which is why *small* clumps are encouraged)

Poly-HEMA Coating of Tissue Culture Plastic

Poly (2-hydroxyethyl methacrylate) (Sigma #P3932)
Cell adhesion is blocked completely on plastic treated with 100 l/ cm2 20 mg/ml poly-HEMA.

kinjal NIPER
kinjal NIPER's picture
 I am working with

 I am working with saccharomyces cerevisiae (liquid suspension media). Now I want adharent cell culture of it.  Can anybody suggest protocol for making adherent cell culture of yeast???