I have seen the protocols for chondrogenesi in a 3D culture (micromass) but I would like to know if there is a protocol for culturing the cells in a monolayer and adding some factors so they can become chondrocytes. Does anyone have a protocol for differentiation and for quantification of the differentiated cells? Thank you very much.
I'm using commercial available differentiation medium according to the manufacture protocols. but you can use this paper as reference http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3698395/
I think that chondrogenic differentiation is better in 3D culture plus TGFbeta1 or TGFbeta3 or BMP2.
I concur that the 3D environment culture is better for chondrocyte differentiation. The pellet culture (or the alginate hydrogel) are the best systems. However, if you see the paper by Kang et al (Tissue Eng Regen Med 10:380-387), they tried the differentiation also on 2D grown cells.
Dear Miriam
I have successfully differentiated hmscs into chondrocytes in 2d. Our results were recently published. You can take a look on our paper. http://www.ncbi.nlm.nih.gov/pubmed/22099022
In general 3d is better however depending on how good are your mscs you can still differentiate them in 2d. My experience suggests that hBM-MSCs differentiate much better in 3d vs 2d, however hair follicle derived mscs can differentiate well in both 3d and 2d.
Good luck,
Panagiotis
Dear Miriam,
2D culture differentiation with hMSCs works just fine with the same chondro mix used for 3D culture,usually with ITS and TGF-beta3.
The advantage with 2D cultures is that you can start with a very low cell plating density(1000/2000 cells/cm.sq.),progress to confluency and induce unlike with the micromass and pellet culture where you start with a higher cell number usually about half a million cells per pellet.
Accumulation of proteoglycan/GAGs can be measured using Alcian blue or DMMB.
Regards
Charan
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