Hi Farhath,

In my experience 3T3-L1 are easy to culture but you might get some headaches when differentiating them. Anyway, don't worry.

I have some questions. First, are those globular cells detached? Are they refringent or opaque? (maybe you could attach a picture of your cells). Have you checked them with trypan-blue?

Usually when cells divide become round, refringent and they may get less attached to the culture flask. If they are detached and opaque I think they are probably dead.

Has this happened with every vial you thawed after freezing the ATCC cells? In that case it could be a freezing/thawing problem.

At how much confluence did you freeze the cells?

Sometimes, to enhance cell survival after thawing I used 20% FBS instead of 10%, until cells were ready for subculturing. Then I continued with 10% FBS.

Please check that your DMEM is high glucose (4.5 g/L). If it is the recommended by ATCC it probably is. I agree with Maria that there shouldn't be very different FBS from CS for growing them (I always used FBS, from a local supplier).

Can you tell me what media and serum you used after receiving cells from vendor and what type of cryomedium you used ?

Hi

In theory it should not affect if you use FBS or calf serum, to perform the expansion.

When you freeze and thaw the cells take time to adapt again, due to the stress they suffer but after several days they adapt and return to proliferate normal.

Hello Rajesh

I just use the normal DMEM recommended by ATCC and the FBS from hyclone. The cryomedium is 95% complete media with 5% DMSO as recommended by ATCC.

Hello Maria,

Thank you for your suggestion. when I first revived the culture after receiving from ATCC the cells looked great with spindle shape. but after the next passage maximum cells are globular in shape and the spindle shaped cells are very few. After the differentiation too the cells are replicating. Do you have any idea about this?

Hi Farhath,

In my experience 3T3-L1 are easy to culture but you might get some headaches when differentiating them. Anyway, don't worry.

I have some questions. First, are those globular cells detached? Are they refringent or opaque? (maybe you could attach a picture of your cells). Have you checked them with trypan-blue?

Usually when cells divide become round, refringent and they may get less attached to the culture flask. If they are detached and opaque I think they are probably dead.

Has this happened with every vial you thawed after freezing the ATCC cells? In that case it could be a freezing/thawing problem.

At how much confluence did you freeze the cells?

Sometimes, to enhance cell survival after thawing I used 20% FBS instead of 10%, until cells were ready for subculturing. Then I continued with 10% FBS.

Please check that your DMEM is high glucose (4.5 g/L). If it is the recommended by ATCC it probably is. I agree with Maria that there shouldn't be very different FBS from CS for growing them (I always used FBS, from a local supplier).

Hello Matias,

I have figured out that the Media and the FBS are working great. But still facing some issues with the cells.

I bought fresh vial of 3T3 cells again from ATCC and tried to grow them. The cells look great during the culture but after the 2nd passage there are some globular cells which grow clumpy on each other along with the fibroblast cells. When I carried the differentiation experiment, the 1st set of experiment worked great and I could see lovely differentiated cells with fat droplets. But 2nd set of experiment with the globular cells looks very fuzzy after differentiation. These cells overgrow on the fibroblast cells from which your unable to see the differentiated 3T3 cells. Is there any possibility to avoid these globular cells Or Is there any reagent to sort only the fibroblast cells to grow in culture?

I am attaching some of the pictures of the cells for your reference.

Please suggest me if you could figure out some solution.

Thank you

Hello Fartah,

thank you for the pictures

Is your lab working with another cell line? I think that you may have contamination with another cell line/type, since these other cells seems to be morphologically different and they grow in foci, in addition to overgrowing your culture.

I don't know how you can get rid of them using reagents, but If possible I suggest using a dedicated set of media and pipettes, etc, for growing 3T3-L1. Do you use antibiotics with 3T3-L1?

Hello Matias,

Thank You for your response.

Yes, we do have different cultures growing in our lab. But I've made sure to use separate hood and incubator for the 3T3-L1 cells.

Yes, I do use antibiotics in my media.

I wonder if you may be getting mycoplasma contamination into the culture. Maybe check for that with a DAPI stain or specific kit.