Hi Elyn,

During my PhD I used the following cocktail to induce chondrogenesis in human BMMSCs: complete media (low glucose DMEM with 10% FBS, l-glut and pen/strep) supplemented with 10ng/ml TGFbeta 1/3, insulin-transferrin-selenium x supplement premix (ITS premix). This was a 21 day protocol, which did not contain dexa, and was always successful as shown using alcian blue and safranin-o tinctorial staining, as well as collagen II and aggrecan expression.I now work on human ADSCs and induce chondrogenesis using a protocol from Guasti L et al., Stem Cells Transl Med, 2012. This protocol contains 0.1uM dexa and 1x ITS (life tech premix).

I hope this helps.

All the best

Dear Elyn,

The chondrogenic media which worked for us is as follows,

With regular changes of media in every three days.....

I hope it works for you.......

Best Wishes...

Thanks a lot Sophie and Purva...

will try both cocktails out soon.

I have actually used commercialised chondrogenic media. However when we ran the sgag analysis and collagen type II content, we did not get the result. Plus we are also trying to troubleshoot getting the cells into the scaffolds and proliferate (the histology that we got was, the cells were mostly located at the surface of the scaffold and not inside the pores!).

Hi Elyn, I just looked at your comment. We found that rotary seeding, rather than static seeding, a more optimal technique  to get the cells seeded throughout the scaffold. 

Dear Elyn, 

the coktail we use in our group is  DMEM (+AbAm) supplemented with Dexamethasone (10-7M), Ascorbic acid-2-phosphate (50ug/ml), rhTGF-B1 (10ng/ml), ITS+ supplement (Insulin 10mg/ml, Transferrin 5.5mg/ml, Selenium 5ug/L, BSA 0.5mg/ml, linoleic acid 4.7ug/ml, Sigma Aldrich) and sodium pyruvate (1mM).

I hope this info is useful for you

Best Regards

I have worked with rabbit pereosteum derived cells. When these are cultured in micromass the following medium was used:

DMAM:F12 (1:1 mix), with 1x ITS, 100 µM ascorbic acid, 100 nM dexamethasone, 40 μg/ml proline (Sigma), 1% antibiotics, 10% serum.

Important to note for you may be that to get chondrogenic differentiation you want the cells to be in a 3D environment, or at least NOT attached in 2D to a stiff substrate. If your material has big pores, do realise that YOU may think it is a 3D scaffold, but if the pores are big enough it is still a 2D environment for your cells. Depending on the size op your pores, seeding the cells in a gel (which could be combined with a porous scaffold)  might be an option, although you may run in diffusion problems. You can also form pellet cultures and combine those with your scaffold (although a homogeneous distribution can be a problem). Alternatively you seed the cells on your scaffold (via perfusion seeding http://www.ncbi.nlm.nih.gov/books/NBK27082/ or other methods http://www.cellogics.de/TisXell.html), but modify the scaffold in such  way that it ptomotes chondrogenic diferentiation. 

Elyn, I developed the in vitro chondrogenesis method. Our first paper provides the recipe for rabbit bone marrow cells: Johnstone et al (1998) Exp Cell Res 238(1):265-72. I read with interest some of the other replies that mention use of serum. We deliberately set out to exclude serum from the medium when we developed it so that you can freely experiment with other manipulations without the interference/complications of serum components. In order to do that you must use all the ingredients in the paper including high glucose medium, which allows all the cells in the pellets to survive and differentiate. With low glucose, the cells in the center often die. Note that the method includes using ITS+ premix, not just ITS - another item that some researchers have since overlooked. We used ascorbate 2-phosphate because that is a stable form of ascorbate that works as effectively as ascorbic acid but has a very short half-life. You neded it in the medium to get good collagen production. Regarding the actual conditions - you do want to be in three dimensions for this to work, with pellet cultures being the easiest method for achieiving that - we chose that method originally to mimic the embryonic condensation such as found in limb formation. However, be aware that you will not get chondrogenic differentiation with every rabbit bone marrow preparation even if you use the exact conditions - there is variability. To maximize your chances you should not passage rabbit marrow cells in monolayer beyond 3 passages after you harvest and plate the marrow - they lose their potential very very quickly after that. Also, the addition of FGF-2 to the initial expansion medium improves proliferation in monolayer while retaining the ability to differentiate, and also seems to improve the subsequent chondrogenesis, However, do NOT include FGF-2 in the actual chondrogenic medium - it inhibits differentiation if used at that stage. We originally used TGF-beta1 and others have since reported TGF-beta3 works better. That is not our experience: in our hands they are equivalent, but it doesn't matter - both work. However, the point made by someone else that you have to be careful with the batches of TGF-beta3 is true, and is also true for TGF-beta1. The activity varies batch-to-batch for both. One other point - do measure actual matrix production - many papers claim to have differentiated cells because they can detect message for collagen II and aggrecan, but cartilage is defined by its matrix and so you should show you get some produced at the protein level. I hope this helps.

Question to Dr. Jorg Fiedler,

Good suugestion. What difference you have noticed between stabilized ascorbic acid and other.

Second, use R&D system recombinant human TGFb3. Is any better than this. Please advise,

Best,

Subhash

There is no intrinsic difference between ascorbic acid and stabilized ascorbate if you add them appropriately. The difference is that the stabilized form requires less additions. It will last in your medium once prepared whereas the ascorbic acid would need to supplemented again due to its short half life. There is no reason to use the non-stabilized form.

Thanks a lot, Dr. Johnstone,

Subhash

thank you very much for all the suggestions. I really appreciate it.

 YOU CAN USE    THE COMMERCIALLY AVAILABLE  INVITROGEN MEDIUM .

Will there be any difference if I were to add ITS and not the  ITS premix?

Since we wanted a completely serum-free medium when we developed the in vitro chondrogenesis system, we used ITS+premix rather than just ITS to provide a source of fatty acids (the '+Premix' we used refers to the inclusion of linoleic acid with BSA as the carrier). If you don't have access to that additive, you could try adding 1% serum instead. Not as 'clean' a system but that doesn't matter depending on what question you are asking.

guys when you are using ascorbate-2-phosphate you might want to look at ALPL, SPP1 and RUNX2 as you may find up regulation of these genes.

I agree that serum-free media would be more appropriate for chondrogenesis of mesenchymal stem cells. We have experimented with serum-free vs. 1% and 10% fetal bovine serum in chondrogenic media, and you can see the results attached. We have used ITS+ and dexamethasone.

Article FBS suppresses TGF‐β1‐induced chondrogenesis in synoviocyte ...

thanks everyone!

Chondrogenic medium was composed of DMEM-HG with 110 μg/mL sodium pyruvate (Invitrogen),10 ng/mL recombinant human Transforming Growth Factor β3 (TGF- β3, Peprotech),10–7Mdexamethasone (Sigma), 200 μM ascorbic acid 2-phosphate (Sigma), 40 μg/mL L-proline (Sigma), 1% Insulin-Transferrin-Selenium-Ethanolamine (ITS-X, Invitrogen) and P/S.

Thanks everyone.