I worked with these cells. I would try the following: 1) split the cells before they are fully confluent; 2) not over-trypsinize cells when passaging them. In their late passages they are indeed growing very slowly or stop growing. Because they are normal (non-cancerous, non-immortalized) cells. So I prepare master stock (2-3 passages after receiving a new lot from ATCC) and working stock (another 2-3 passages). I do not use then these cells beyond 20 passages

Dear Kurochkin,

Thanks for your comment.

The problem is I don't know the exact PDL of these cells. But I guess they aren't senescence, since the morphology of attached cells is very nice and look like the pictures which ATCC provided in their website (However I didn't buy my cells from ATCC).

You can try to use MEM for your cell

What about Sodium Pyruvate? How manny cells do you seed per cm^2? Maybe the growth area is already too small and you have to increase it instead of decreasing. Also, like Zhaolin Su said try MEM since it is the recommended media by ATCC.

Dear su,

DMEM media is so much richer than MEM or EMEM. It contains two to four times as much glucose and amino acids. It also contains Iron.

Hi Ramin

Considering that the cells you are using are not in the phase of late passage, you might check the pH of the growth media. It is highly recommended that only normal glucose-DMEM should be used for culturing MRC5 cells.

As the pH of the media is most crucial for maintaining nutritional integrity and oxygenation level, I would recommend to  check it once. Also, in order to ensure the stability of media throughout the culture process, I would recommend to put 2-3 ml of media in a growth flask/dish in the incubator without any cells in it. It will serve you two purposes. Firstly, it will ensure that there is no contamination in the culture throughout. Secondly, the color of media in the incubator will ensure that the pH is stable and the media is good enough for growth. 

You need not further increase the FBS concentration. Rather, 10-15 % is more than good. High glucose content is also not necessary. You can try the general DMEM such as Sigma D6046. 

Best of luck:)

Dear Szymanski,

My DMEM contains Sodium Pyruvate and the I seeded about 20000 cells/cm2.

Although MEM recommended by ATCC, the repository which I bought my cells from them recommended DMEM (High glucose) which is richer in all aspects than MEM or EMEM.

Dear raman,

We culture MRC5 cells. Mr. Kurochkin answer is appropriate to follow.  Even though cells seems to be slow growing their growth continues as it is. FBS 10%, MEM(1gr glucose)/DMEM(4.5gr glucose)/RPMI(2gr glucose) with sodium pyruvate without NEAA works well. If you find morphology as per ATCC and with out contamination you are doing well. Split after every 3-4days once not  immediate or alternative days which gives more trypsinization stress to them. Accept the cell growth as it is. Try to plan your experiments according to their growth rate. I don't suggest to change composition rather than ATCC prescribed one.

Dear Shekhar Dasari,

Thanks for your comment. Why do you prefer no to use NEAA ? As I think NEAA could enhance growth rate of many cell lines.

You suggested split after 3-4 days, but after 3-4 days there isn't any change in my cells confluence. What is the doubling time of your cells?

Again thanks for your help.

Dear Raman,

Excuse me for delay.

1)Addition of NEA is not always mandatory to cells. Cancer cells require lots of nutrients which can produce instant energy. But normal cells/ even immortalized depends on regular oxidative phosphorylation and other savage pathways like gluconeogenesis for their growth. Providing more nutrients is  not recommended to these cells since they alter cells regular mechanisms(literature). But for cancer cells increased proliferation and metastasis observed(literature). MRC-5 cells are can grow in MEM well as DMEM. I did not observe much difference in their growth rate.

2)Trypsinization effect and frequent pH variation can be avoided for slow growing cells. So that they wont under go differentiation which retard their growth.

3)Our cells doubling time is 24-48 hrs.

Its my pleasure to share my experience.

Dear CHANDRA,

Thanks for your follow-up. MEM isn't available in our lab but we have DMEM, alpha-MEM and RPMI. I'm going to change my basal media to RPMI-1640 because of it's similar osmolality to EMEM which recommanded by ATCC and ECACC.

My cells doubling time is more than 72 or even 96 hours. In Iran there isn't any other repository for this line and now I'm completely confused !!!

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