Dear Ena,

If your goal is chondrogenic differentiation,i would suggest that you could use FGF-2(2ng/ml). Using FGF-2 in the culture is known to enhance chondrogenic differentiation. Plenty of literature is available. I've attached one of them.

Best,

Charan

Question to Charan:

how many weeks chondrogenesis should be terminated to avoid hypertrophy for BM-MSCs

Dear Sir,

I have seen that the chondrogenic markers peak at 8-16 days in culture(Col2A1 and Aggrecan)Post 16 days of differentiation there could be up-regulation of the hypertrophic markers like col X..I believe that at day 21-28 there is definite hypertrophy.

Regards

Charan

Hello 

Dulbecco’s Modified Eagle’s Medium, High (4.5 g/l) Glucose (DMEM-HG, Invitrogen) supplemented with 10% ITS+ Premix Tissue Culture Supplement (Becton Dickinson), 10−7 M dexamethasone (Sigma), 1 μM ascorbate-2-phosphate (Wako, Richmond, VA), 1% sodium pyruvate (Invitrogen), and 10 ng/ml transforming growth factor-beta 1 (TGF-β1, Peprotech, Rocky Hill, NJ).

for Chondrogenic Differentiation Protocol :

1. Plate 1 x 105 MSC per well in a 96-well

U-bottom suspension culture plate using

MSC Growth Medium (DMEM-HG). Work

in duplicate.

2. Spheroids will spontaneously form

within 24 - 48 hours.

Note: The more cells are used, the bigger

the spheroids (up to 3 x 105 cells per

well).

3. Induce one of the duplicate samples

with MSC Chondrogenic Differentiation

Medium (DMEM-HG). Use MSC Growth

Medium for the remaining well as a

negative control.

4. Incubate for 21 days. Change Medium

every third day. Be careful not to aspirate

the spheroids.

5. Fix and stain the cells for chondrogenic

markers.

good luck

Thank you all! Our differentiation medium is similar to what Saleh described, and for expansion we currently add FGF1 to DMEM. It seems these are what work best for most people.

Thanks again,

Ena

For BM-MSC expansion without differentiation, I highly recommend StemCell Technologies Mesencult basal media with their Stimulatory Supplements.

Dear Ena,

we compared four different kinds of media to expand MSC. MesenCult works best to our experience. MSC show a very high proliferation and poulation doubling time is reduced about 50 % in comparison to DMEM+FCS. Unfortunately, MesenCult is very pricey. We use it for expansion of MSC directly from bone marrow samples, and after the first passage we acclimatize the cells to DMEM (to reduce costs). At least I state that I am not related to Stem Cell Technologies.

Best regards

Dirk

Hi Ena, 

For expansion of MSC we use LifeTechnologies Keratinocyte SFM with 5% FBS, 2mM of N-acetyl-L-cysteine and 0.2 mM of L-ascorbic acid 2-phosphate and it really works great with fast population doubling times. For differentiation we use Stempro medium from LifeTechnologies.

Best

Maciej

If you use any expansion media that is not specific for MSC (such as Mesencult) addition of FGF2 will greatly help your expansion. Just as crucially, you test to test the serum you use. Either buy MSC qualified serum (expensive) or you have to test serum batches yourself to ensure chondrogenesis is maintained. Most serum batches do not maintain MSC multipotency very well after proliferation.

For the hypertrophy question, terminating the experiment early doesn't avoid hypertrophy, it jiust means you don't see it. Thats two different experimental outcomes.