Ideally, it is better not to exceed 24 hours while generating conditioned media. You can either centrifuge it between 5000 to 10,000 rpm for 10 minutes at four degrees or filter it with 0.2 micron syringe filter. Do not use 100% conditioned media. It will kill your cells. You have to titrate it for optimal volume, for example 10% conditioned medium and 90% culture medium; 20% conditioned medium and 80% culture medium, 30% conditioned medium and 70% culture medium and 40% conditioned and 60% culture medium. It is not a good idea to exceed 40% conditioned medium.

One important problem that has been overlooked is appropriate controls. For example, if you use 30% conditioned medium and 70% culture medium, what would be your 'control' medium? The best option I could come with is to incubate the medium alone without cells for 24 hours (or 48 hours) and use this as control. For example 30% of this medium and 70% of culture medium.

If any one has any better suggestion for 'control' medium feel free to post your suggestions.

I condition media for at least 24h but no longer than 48h starting from the moment that the adherent cells restore their regular morphology after passage. It depends how fast your cells metabolize - do not allow the phenol red in the medium to get orange. In case of using the medium for different cell line I would recommend filtering to avoid contamination (in case adherent cells float it does not mean they are dead). I usually add the conditioned medium to up to 40%. Working in different lab I was using conditioned medium prepared on stimulated PBMCs, and it was OK to keep it frozen at -20 for up to 2 weeks (no one checked longer, probably at -80 could stand longer).

Like Marcin, I have done the conditioning for no longer than 48h. In the experiments I did, I did not use 100% of the conditoned media, but diluted it into fresh media, with a maximal concentration of 50%. to remove any cells/cell debris I centrifuged the conditioned media. Whether you can freeze the media depends on the kind of factor in it that you want to study. Some growth factors and cytokines do not take kindly to freezing and thawing.

I routinely generate conditioned media from L6 myotubes that I treat overnight. When I collect the media, I don't filter it, but I centrifuge (5,000 g for 10 min) to get rid of cells and debris. Filtering through 0.2µm works too.

When I treat other cell types with it, I use it right away without diluting in fresh media, but I guess you could dilute it as Marcin suggested if your effect is strong enough. I noticed a significant reduction in glucose in conditioned media compared to fresh media, so think carefully of the controls you need.

For storage, I keep it at -80°C and from my experience it is stable for months. Just avoid freeze/thaw cycles.

Ideally, it is better not to exceed 24 hours while generating conditioned media. You can either centrifuge it between 5000 to 10,000 rpm for 10 minutes at four degrees or filter it with 0.2 micron syringe filter. Do not use 100% conditioned media. It will kill your cells. You have to titrate it for optimal volume, for example 10% conditioned medium and 90% culture medium; 20% conditioned medium and 80% culture medium, 30% conditioned medium and 70% culture medium and 40% conditioned and 60% culture medium. It is not a good idea to exceed 40% conditioned medium.

One important problem that has been overlooked is appropriate controls. For example, if you use 30% conditioned medium and 70% culture medium, what would be your 'control' medium? The best option I could come with is to incubate the medium alone without cells for 24 hours (or 48 hours) and use this as control. For example 30% of this medium and 70% of culture medium.

If any one has any better suggestion for 'control' medium feel free to post your suggestions.

I am also performing some experiments with the conditioned medium. After collecting the conditioned medium I centrifuge at 5000 rpm, collect the supernatant, filter using 0.2 micron and store at -80oC. Storage up to 2 weeks had no issues in my experimental conditions.

I use 1:1 ratio of condition medium:fresh medium. All my experiments are not more 48 h.

I use CM from mesenchymal stem cells for various assays.I centrifuge the media as well as do a 0.22 micron syringe filtration.Store at -80 till utilized.

I would suggest you to dilute the conditioned media in serum free media to 50% to look at the effect of the cytokines released by your cells.You can try various gradient dilutions.100% CM could be toxic.

I agree with Above scientists. C. Morais has raised a good point regarding controls. The control suggested by C. Morais is a good idea. Here are 2 other controls.

1. Since Frederica is looking for effects of chondrocyte CM on the osteoblasts, its worth trying CM from simple fibroblasts..WHY? because chondrocytes differentiate from fibroblasts, and control CM from fibroblasts may help subtract out less interesting extracellular factors.

2. Many chondrocyte cell lines lack the original markers and growth properties of primary chondrocytes.. so it would be useful to use CM from primary chondrocyte cultures as another control. In fact, CM from primary chondrocytes may have very interesting effects on osteoblasts. Ofcourse, it is more difficult to reproduce data with primary chondrocytes...because they dedifferentiate rapidly in culture (within 1 week). Still, it maybe worth trying. Hope this help-Good Luck!

Hi, Federica, the usage of conditioned media (CM) is largley dependent from the aim of your experiments. I completely agree with prevuous comments that you should use 50% or even 30% of conditioned medium only. And from the cell which cultured not more than 24 hours. The main reason of this restriction is: cell were secreted to the medium a couple of growth factors like cytokines during exponetial cells growth, However, after longer cultivation (after exponetial phase) some toxic compounda may also appeared. (you may observed this as change in phenlo-red color, ea. pH). So, in fast growing cells culture you should collect conditioned medium after 20-30 hours.

Once you collect conditioned medium, you can filtered it through 0.2 µm filter and keep at -20 C for up to few weeks.

Please, consider also that CM is a some kind of "black box" and you never know which growth factors were realised by fider cell and what was the concentration.. Many small molecules may also realize in the medium and also have effect on the cell growth..

Ideally, it will be nice, of course, to perform HPLC or MS analysis of CM to know which molecules have a stimulative role..

Good luck!

Hi, Federica, in addition to previous post, be sure that you collect CM during middle stage of exponential growth. If you collect CM during stationary phase, you will have inhibitory effect.

I was wondering what could happen if you incubate your primary chondrocytes in the media derived from primary osteoblast??Could they form hypertrophic chondrocytes...push them towards osteoblastic lineage??Any ideas??

Hi, Charan, I think it is possible that fider cells were realized some growth factors which can change cell fate. However, it will be not easy to indentified such factor because CM is a "black box" and results may not be 100% reproducible..

1. Conditioned media will have the following vital components depleted (including but not limitted to) glucose, glutamine, serum

2. Conditioned media will have the following components secreted/excreted by cells (including but not limitted to), specific cytokines, chemokines, lactate, glutamate

While people are interested in certain conditioned media for the secreted cytokines/chemokines, the depleted vital components and secreted/excreted components are the limitting factors that can induce stress in the experimental cells

These issues are solved by dilution with complete fresh medium. 1:1 dilution is the best because it can provide glucose and glutamate to support cells for at least 24 hours.

I would strongly recommend 0.2um filtering than centrifugation to avoid cross contamination of cells

For appropriate controls and dilutions, filtering see

https://www.researchgate.net/publication/233710524_Smac_mimetic_enables_the_anticancer_action_of_BCG-stimulated_neutrophils_through_TNF-_but_not_through_TRAIL_and_FasL?ev=prf_pub

Article Smac mimetic enables the anticancer action of BCG-stimulated...

Dear All,

you All raised some very good points. I wish I could use primary cells as controls, especially primary chondrocytes, osteoblasts and, my favourite, synovial fibroblasts. I use to produce beautiful primary cultures in the past, in a previous department, but at the moment I have only cell lines available. This, on one end ,should allow to obtain more reproducible results, on the other end I will of course lose some of the carachteristic of the original cells seen that I am using basic immortalised lines... a chondrocyte cell line will never have the same behaviour of a primary chondrocyte, fair enough. However I feel there is still a lot to discover in the pathways and the ways of "cross talking" between the tissues of a joint and these cell lines can represent a starting point. I hope to have access soon to surgical waste (I am in the process of obtaining the ethics) and produce primary lines so to start the conditioning process, I would also try that in bioreactors!

So to make a summary of your suggestions:

-filtering for sure with a 0.22um filter. Some of you also couple centrifugation. I may try both.

-collection of the media from the "donor" cells during the exponential growth phase

-conditioning for no longer than 48h

-dilution of the conditioned media in a ratio 1:1 with fresh culture media (maybe woth trying a 1:4 also, according to some suggestions)

-ok for freezing (-20 or -80C), no freeze/thawing.

Thank you All!

Dear All,

All the suggestions were really valuable and one should keep in mind while collecting the conditioned medium and using it for cell based assays.

As Taras Pasternak mentioned conditioned medium is a black box. In order to get reproducible results one should first understand and characterize the secretome and then concentrate and use only the particular fraction in order to get maximum efficacy.

Hi Anais,

I am the none that originally asked the question.

I've been through several experiment with the use of conditioned media now, so I can give you some info.

It is important you collect medium from the logarithmic phase, so when your cells are still actively growing (so I would say around 70% of confluence). It is important you keep consistency between seeding densities and collection, making sure the medium is always collected from cells at the same density. A good idea would be to freeze the medium into aliquots for multiple future experiments, or after you collected from different passages, pool it and freezing it in working aliquots.

Hope this helps.

All the best

Federica

Yes, Frederica's experience is good to remember. These points apply to all cell culture experiments. We tend to compare experiments when starting variables (passage number, seeding densities, log/non-log phase growth) were different . This leads to non-reproducible or weird results.

Query for Frederica:

Have you tried using specific ECM for the osteoblast culture? This will give more "in vivo" type of data, and the CM you collect can give you an idea of stromal-osteoblast interaction. If not ECM, you can do co-culture with the synovial fibroblasts you mentioned. CM from this co-culture would also give useful data--correct?

Dear All,

I am going to prepare a stem cells conditioned media..I was wondering how long do you incubate the cells before you filter it?because mostly I found the reference they incubate it for 3 days then they filter the media..so I already optimize my cell density for the CM..but recently I found a reference that only do a day..

Hey Guys,

I have produced TCM cells and wanted to ask, wether I can store it at 4°C when I use it within one week. I want to do a kinetics and have to supplement the cells every 48h with "fresh" TCM. By storing it at 4°C I wanted to avoid the freezing and thawing stuff. Can I do it that way?

Thanks in advance.

Nice to know all about the collecting and applying conditioned medium. I would like to know one more thing about the initial medium to be used for the preparation of CM. Is it necessary to add some percentage of serum and antibiotics in the initial medium or not?? I would be happy to know about. I just use serum free medium without any antibiotics.

I think conditioning is not so straight forward. It depends on the experiments you are doing and what you are trying to obtain from the conditioned media. The media being conditioned will always have less nutrients than fresh media as it gets consumed by the conditioning cells so it may be important to control for that. Either serum or growth factors are required to obtain the right secretory factors from the conditioning cells because it will effect the conditioning cell's proliferation and signalling. I would try to avoid the use of antibiotics as they are not necessary for cell culture.

Hi Sonia,

I really doubt that the endothelial cells would still be adherent while secreting growth factors into the media at 24 hours in serum free media. You could use a small amount of serum while you are collecting the conditioned medium. Maybe you could use 1-2% serum and use a 1-2% serum containing medium as your control for the experiments.

Regards

Charan

Hi eveybody,

   I was wondering if any of you ever tried to run a Western Blot or ELISA to detect levels of cytokines in Conditioned Medium? Do you need to concentrate it or you can still use it as it is? What did you use as loading control for Western? 

Hi Salvatore,

Forget the Westernblot, not sensitive enough and tricky to get peoperly quantitative. I've done plenty of ELISAs for cytokines from conditioned medium, and never needed to  concentrate the CM. Quite often it actually needed diluting.

best wishes,

Rob

I completely agree to what Rob said. ELISA would be the best way to estimate cytokines/growth factors in the CM. Mostly dilution of the CM is what is required for most growth factors for them to fall within the range of standards provided. However, to detect some factors secreted in lower amounts, concentration using 1 KDa or 3 KDa ultra-centrifugation. 

Good luck!

To be very concise, I take the conditioned media and filter it to remove any possibly suspended cells or etc and then dilute it with the fresh media (+10%FBS) in 2:1 ratio. And indeed it is working for my application with the neonatal cardiomyocytes.

Just wondering, when you generate your conditioned media, how much media do you add (let's say in a 6 well plate for 24 hours)?

All of you say to never use 100% of the conditioned media because it's toxic, but a post doc told me they've always used 100% and their cells were fine.

Also I'm not sure whether to filter or centrifuge my conditioned media. Filtering sounds cleaner, but is it possible that when you filter, you may remove or reduce the number of cytokines?

Hello,

Many of you suggested that 100% conditioned media would be toxic. I am working in models where 100% conditioned medium is toxic for cell x but safe for cell y. Although I can imagine many mechanisms by which the conditioned media might be toxic, as anyone identified which molecules in the conditioned media are toxic using an unbiased approach?

A good control would be media without CM and media plus CM from 80% confluent cells (1:1 or whatever you can use). The different level of confluency plus the no CM control should help data interpretation.

Would you please, any one give me suggestion about preparing conditioned media? I want to check Par 4 secretion. I need the protocol to prepare conditioned media.

Would anyone please give suggestions about the concentration of serum for the donor cells?