Hello Everyone,
I am currently trying to differentiate pre-adipocytes isolated from mouse adipose tissue to matured adipocytes in culture. Until now, I get about 70% differentiation and will like to have 100%.
How do I ensure that the cells I had in culture prior to induction/differentiation are all preadipocytes?
How do i achieve 100% differentiation
When the adipose tissue is digested I suggest you to put the 50 ml conical tube in ice for 20 minutes. The mature adipocytes will float and you can remove them, the top layers (I would say the 2/3 on top) will contain the pre-adipocytes you can now differentiate. Cold separation will help you to purify the preparation. To improve differentiation you could increase the concentration of insulin you use in your medium and also adding some rosiglitazone could help.
You can also maintain cells in insulin supplemented media (5ug/mL) post differentiation. The longer you keep them in culture the greater the lipid accumulation will occur. To ensure you are isolating the pre-adipocyte fraction, take your collagenased tissue and perform centrifugation at 1000xg for 3 min and you will see a nice pellet which contains your "pre-adipocytes" and only these cells will adhere to your tissue culture plate.
Hi,
first of all, can you share your differentiation culture medium composition and your cell isolation and culture methods. It will be easier to help you to improve your differentiation.
I guess you put rosiglitazone or other PPARg agonist into your medium to reach 70% of differentiation. You have to take into account that not only progenitor cells stick into your plastic dish. Macrophages and endothelial cells could represent more than 30% of your cells depending upon obesity status of your mice. Anyway if you are working with freshly isolated cells (without extensive passages) you obtained cells in different state regarding adipogenic process. Using Immunoselection or Facs to isolate cells based on described markers (Sca1+/CD45-/CD31- and potentially PDGFRa+) could help you to improve the purity of the cells you get.
I am not a big fan of increasing Insulin concentration since the limiting factor could become the glucose concentration into your medium.
Hi David,
Thank you for your help.
Cell culture condition
Cells are proliferated in culture media; DMEM/F-12 GlutaMAXä (Invitrogen) containing 15% FBS (Gemini Bio-Products, Benchmarkä), penicillin/streptomycin and Hepes buffer 2 mM (Invitrogen, Cal# 15630).
Induction media;
DMEM/F-12 GlutaMAX (Invitrogen, Cal# 10565-042) containing dexamethasone (5 M, Sigma, Cal# D-4902), insulin (0.5 gml-1, Sigma, Cal#I-5500), isobutylmethylxanthine (0.5mM, Sigma, Cal#I-7018), rosiglitazone (1M, Cayman, Cal#71740), T3 (1nM, Sigma, Cal#T-2877) and 10% FBS (Gemini Bio-products, Benchmark Cal# 100-106).
Maintenance mdie:
media containing insulin (0.5 gml-1), T3 (1nM) and 10% FBS until they are ready for collection
The SVF was collected by digesting the adipose tissue with collagenase and dispase. DMEM was added to stop the digestion after shaking in water bath at 37oC. The sample was filtered through 100um strainer, centrifuged, and then filtered through 40um strainer and centrifuged again. The pellet was then suspended in the culture media. After 4 hrs in culture the medium was aspirated and the attached cells were washed with PBS (twice). After this wash step the cells were incubated in the culture media.
Almost confluence cells were incubated with induction media for 4 days (media was changed every 48 hrs). The cells well subsequently incubated in the maintenance medium for 6 - 8 days
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