Hi everyone,
I have been struggling with the differentiation of mouse SVC cells into mature adipocytes lately.
In the past, I have always isolated and cultured SVC in 15% FBS HG-DMEM and induced differentiation using an MDI cocktail (no rosiglitazone added) (0.5mM IBMX, 2uM Dexamethasone, 10ug/ml Insulin), the result was always good with ~ 80-90% cells showed adipocyte phenotype, and lipid droplet formation.
However, recently, all of my cells are not able to differentiate anymore, regardless of all of my attempts:
1. Younger mice: I usually use 3 weeks - 5 weeks old male mice
2. Refresh of chemical stock: I have refreshed and bought all of the MDI components
3. Seeding: I've tried seeding on all 6 wells, 12 wells, and 24 wells plates with low and high confluency
4. Adjust FBS level: although my previous results were just fine with 15% FBS, I have tried 5% and 10% after having the problem but it was not solved
I have always had a good amount of viable cells after isolation following this protocol:
https://star-protocols.cell.com/protocols/552
I am searching for help and suggestions, please leave a comment if you have any experience with the same issues or if you have some solutions for it.
Thank you all!!!
Stromal vascular cells (SVCs) are a heterogeneous population of cells found in adipose tissue, including preadipocytes, endothelial cells, fibroblasts, and immune cells. Adipogenic differentiation of SVCs refers to the process by which these precursor cells differentiate into mature adipocytes, capable of storing lipids. Here's a general protocol for inducing adipogenic differentiation in stromal vascular cells:
Materials Needed:
For those who have the same problem, I have just figured out that without any enrichment step after SVF pellet collection, other cell types can easily retain in the SVF mixture and competitively grow in the culture plate.
For some reason adipocyte progenitor populations of SVF attach faster and do not require a long time, hence, removing and changing to the fresh culture medium immediately 2 hours after seeding seems to reduce the attachment of other cell types. This adjustment significantly improves the differentiation induction in the downstream experiments in my case.
Hope this helps you all~!