Hi Binal,

by specks if you mean floating debri, there are few things you can try-

1. Change the trypsin 

2. Reduce the time for trypsin action

3. Post trypsinization wash your cells with DPBS or plain RPMI media.

Also- a T25 culture flask when confluent has approximately 2 million HepG2 cells and a T 75 culture flask has between 6-7 million cells.

For Si RNA treatment follow the company's instruction for seeding cell. Generally they try a low seed for better treatment.

Hope this works and Good Luck to you!

Shilpa

Hi Binal, do you find debris during culture or only after trypsinization?

Depending on cell confluence we obtain 15-18 million cells when cultured in T75 flasks.

For siRNA treatment I agree with Shilpa, you should follow company's instruction for seeding cells.

Hi Binal,

As the previous posters have said, it appears to be nothing more than cellular debris from the tripsinization process; nothing major to be worried about as this is quite common.  It is almost impossible to remove debris entirely, even with multiple washes.

Just an additional observation, if you want to grow your cells a bit faster, up the seeding density a bit. 

As has been said by the previous posters is right to limit the confluence in T75 culture flasks to about 6 million cells and to trypsinize for no more than 1 minute. Some debris are always present after trypsinization and must be removed by washing.

We usually use DMEM+10% FBS for HepG2 cells. Regarding debis, do not use high speed centrifugation (about 1000x). Check what you cells do not like--it might be FBS you use. However, if this debris is a constant event, better find the cells from another source, these "unhappy" cells become less sensitive to the treatments and transfections.   Plating density for 6-wells is about 4X10 5.

I agree with what everyone else has mentioned.

Make a list of the different recommendations, and try till you find the condition which makes your cells happy. They definitely look unhappy.

This cells will do well in either FBS (preferable if you can afford) or BCS (affordable).

avoid trypsinizing for longer than a minute.

DMEM or MEM media are equally great but you want to be consistent with your media of choice.

Please if you don't mind, I will like to know the contents of your media besides the serum and the media itself.

Hi Binal:

Sometimes these debris are due to heavy mycoplasma contamination. Do you periodically control your cells for mycoplasma contamination?

Hi Binal,

You should respect the manufacturer instructions for HepG2 cells culture.

We also use DMEM+10% FBS and 4X10 5 plating  density for 6-wells plates. Small amounts of small cell debris are usual, even efter washing 1 time.

Important : never reach confluence with these cells (max 80-90% of confluence; then the trypsinization time should be reduced to  1-3 minutes). Otherwise cells become more difficult to trypsinate and you will get more debris.

Moreover we observed that their metabolism could be different if overloading the seeding or exceeding the confluence state.

Good luck

Hi Binal

For my previous experience with HepG2 there are 2 things that might be important besides what has been said previously. When we thaw a new vial of HepG2 cells we usually cover the plate with a 1:10 collagen R solution for 5 minutes and then just collect the solution after and this can be reused often. This is just to help the cells re-attach after thawing and when you subculture them you don't need to use the collagen. We saw that this procedure is important when you have some old vials of cells (after 30 passages). 

Another thing that happened to us was some changes in organelle morphology when we changed the trypsin we were using and we believe that was due to an excess of EDTA, so check for the composition of your trypsin to see. Right now we are using the trypsin from gibco and we don't have problems.

Hope it was useful

Mónica

Thank you all for your responses, we have decided to use EMEM(ATCC recommended) and 0.05% Trypsin 0.53 mM EDTA. I am going to thaw a new vial soon.

Monica - Can you explain in detail regarding using collagen R solution, along with catalogue number for the same? Do you just add the collagen on the plate or T-75, (is it tissue culture treated or non-treated) and then remove the solution, and then add the thawed cells along with media on top? I do not know the passages of these old vials, it says P6, and it was originally cultured in RPMI.

Hi Binal

The Collagen solution is quite easy. We order Collagen R Solution 0.4%, from Serva, ref. 47255.01, here is the link if you want more info on the product, but it can be another brand.

From this, we make a 1:10 dilution in ddH2O and then sterilize it by filtration. Just add enough of solution to cover your new plate or flask and let it stay for 3 to 5 minutes. After, just recollect the solution so you can use it again, and fill your plate/flask with medium.  For us we only need to treat the plate when we thaw the cells and in 1st passage to another plate, afterwards they attache nicely without help. We usually reuse the solution up to 25/30 usages, but you can try to use it longer.

Best regards

Mónica

http://www.serva.de/enDE/ProductDetails/247_47256_Collagen_R_solution_0_4_percent_sterile.html

Hi Monica,

The Collagen treatment worked and cells now adhere.

Thank you,

Regards,

Binal