This is an interesting question, which I am also trying to get a clear answer on.
In the 1980's people observed that chondrocytes lose their phenotype rapidly in monolayer culture. As a consquence in the early 1990's protocols were developed to counteract this; alginate encapsulation, micromasses and pellet cultures (3D culture methods). This type of 3D culturing has become the standard in (articular) chondrocyte research. It is interesting that the initial papers do not routinely compare monolayer to 3D culture techniques.
In the 1990's people startedusing MSCs and discovered their multipotency in vitro. I guess they employed the same 3D culture techniques as the chondrocyte researchers to achieve chondrogenesis (I am still trying to find the first paper where this was done). In this context it is usefull to mention that chondrocytes that have lost their phenotype in monolayer, re-acquire their chondrocyte phenotype in 3D with the correct growth factor cocktail (tentatively called re-differentiation).
The reason that MSC can also undergo chondrogenic differentiation in monolayer is probably because they form their own 3D environment. Usually monolayer differentiation is performed at high cell density and the wells get overconfluent quickly, if not immediately. Subsequently they start growing on top of each other, creating some kind of 3D environment and later express COL2A1 and ACAN.
Dear Akshata,
Chondrogenic differentiation of mesenchymal stem cells is also done as a monolayer as well as as micromass culture differentiation apart from pellet culture.
MSC pellet culture differentiated chondroblast mass characteristics closely mimic the cartilage.
When MSCs are differentiated in a pellet culture system,they first condense resulting in growth arrest,formation of pre-chondroblasts and adaptation to high cell density(day 2 to day 10).Then begins the proliferation of chondroblasts resulting in matrix deposition and then terminal differentiation with further more matrix deposition(Day 10 to Day 28).
Hi colleagues,
interestingly, Fujinaga et al reported that spontaneous chondrogenic differentiation of bovine MSCs in pellet culture even occurred without addition of any external bioactive stimulators, namely factors from TGF-beta family, which were previously considered necessary (see Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells in pellet cultural system. Exp Hematol. 2004 May;32(5):502-9)...
Could someone describe a micromass or monolayer differentiation protocol for this? I am interested in exploring alternative ways of assessing chondrogenic differentiation in human MSCs. Thanks!
Dear Athena Russell,
If you are looking out for protocols then please have a look at following paper. here they have followed micromass culture method for chondrogenic differentiation.( this protocol worked for us).
Kind Regards,
Akshata.
Comparison of proliferative and multilineage differentiation potentials of cord matrix, cord blood, and bone marrow mesenchymal stem cells
Prathibha Shetty, Khushnuma Cooper, and Chandra Viswanathan
One thing to monitor when using chondrogenic pellet culture is the level of hypertrophy. While there are many chondrogenic genes upregulated, it is generally accpeted, especially in the case of bone marrow derived cells, that the cells undergoe endochondral ossification. For resting articular cartilage this is not the pathway you want. Longer term culture can also lead to necrosis in the center of the pellet.
Dear Dr.Martin,
In the context of the pellet culture system of human mesenchymal stem cells,on which approximate day post induction could the hypertrophic genes say col X upregulate?Do you think there may be actual hypertrophy in the pellet at 21 days(a general protocol) or do they upregulate with extended differentiation?
It would be really helpful if i could get an answer with regards to your experience.
Regards
Charan
Stempeutics Research
Dear Charan
markers of hypertroph, such as collagen x, are often visible within 7 days.
Hello,
MSCs can also be induced to differentiate into chondrocytes using alginate culture. The cells also go through stages of cell cycle arrest and differentiation. Here is the article: http://www.mc.ntu.edu.tw/department/anatomy/chen%20Yuh%20Lien/Bata_paper/26-Chondrogenesis%20of%20human.pdf
Dear Athena,
There are many protocols for MSC to chondro differentiation in monolayers - search in PubMed for solution that suits you best ( e.g. depending what kind of growth factors you have). I'd suggest differentiation in monolayers beacause as Dr Martin said in pellet culture there is bigger risk of osteo/chondro diff.
I still haven't found a really good detailed monolayer differentiation protocol for chondrogenesis.
How about these?:
- Effects of in vitro low oxygen tension preconditioning of adipose stromal cells on their in vivo chondrogenic potential: application in cartilage tissue
- Molecular and cellular characterization during chondrogenic differentiation of adipose tissue-derived stromal cells in vitro and cartilage formation in vivo
This is an interesting question, which I am also trying to get a clear answer on.
In the 1980's people observed that chondrocytes lose their phenotype rapidly in monolayer culture. As a consquence in the early 1990's protocols were developed to counteract this; alginate encapsulation, micromasses and pellet cultures (3D culture methods). This type of 3D culturing has become the standard in (articular) chondrocyte research. It is interesting that the initial papers do not routinely compare monolayer to 3D culture techniques.
In the 1990's people startedusing MSCs and discovered their multipotency in vitro. I guess they employed the same 3D culture techniques as the chondrocyte researchers to achieve chondrogenesis (I am still trying to find the first paper where this was done). In this context it is usefull to mention that chondrocytes that have lost their phenotype in monolayer, re-acquire their chondrocyte phenotype in 3D with the correct growth factor cocktail (tentatively called re-differentiation).
The reason that MSC can also undergo chondrogenic differentiation in monolayer is probably because they form their own 3D environment. Usually monolayer differentiation is performed at high cell density and the wells get overconfluent quickly, if not immediately. Subsequently they start growing on top of each other, creating some kind of 3D environment and later express COL2A1 and ACAN.
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