Mainly, it depend of the capability of your cells to differentiate to chondrocytes. When they do micromass, they have the capability of differentiation.
Dear Dr. Roche,
Please elaborate your answer in detail. Differentiation takes place in both. Please educate me about clear difference between two, I became interested too as Mr. Pham has raised the question.
Thanks,
Subhash
You need to determine what is a differentiation into chondrocyte, just the presence of COL2A1 mRNA or the production of COL2 protein, glycosaminoglycan (GAG) and proteoglycan. In addition, you could want to observe a specific cell morphology, with a round shape.
I think that we need these parameters to define a chondrocyte and the production of a cartilage like tissue. I think that GAG are the major extracellular molecule of the cartilage. In this way, micromass is an important step, with production of GAG, the good shape of cell and a production of type 2 collagen.
I have seen this happen if you increase the number of MSC to more than 1.5 mil per pellet. In lower numbers they should form a pellet even if they do not differentiate well into chondrocytes. If you use 15ml falcons they can look donut like sometimes because of the small plastic protrusion at the bottom of the tube. Depending on donor it can happen within 24h or take a couple of days for the pellet to form. Also if you do not add FBS/FCS to your pellet culture it can reduce the capacity of your MSCs to produce pellets.
However not forming pellets does not mean you will not get any differentiation. Continue with your culture try not to move them a lot and be careful when you change your media (always leave some behind so that you do not disrupt them) and if you are getting any chondrogenic differentiation they should be more robust after 7-10 days and they should start increasing in size and becoming more 'bouncy'.
Then you can check them by gene expression levels GAG and histology as it has been suggested by others before
Good luck
Let the MSCs first aggregate 1 to 3 days in serum-containing growth medium. Start the chondrogenic differentiation after a stable micromass has formed. Centrifugation after seeding will aid micromass formation.
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