I grew these cell lines for a long time periods. You can try grow them in 20% FCS

Hi Prahlad:

The widely used/accepted culture medium for HMCLs is RPMI + 5-10%FBS. First of all check your FBS (batch nr, company) and whether it's used for other cell lines in your lab. I have been using these HMCLs for a long time.I don't know what your treatments are, but they may behave differently in some ways, mostly depending really on how you want to treat them. If the viability is OK and also the number of passages is reasonable (say, no more than 15, as I believe) then you should think about your experimental treatments, is that a drug?, cytokine?, a specific ligand?, etc.

In my experience, RPMI 8226 is really easy to handle and culture, U266 displays some kind of resistance to some treatments (e.g. some apoptosis-inducting agents), OPM-2 is among others more sensitive to treatments (in my experience, bortezomib and doxorubicin). Also you need to know if your treatments require some kind of "receptor-mediated"processes, because expression and functionality of some receptors may change over time (of culture maintenance).

Hi Jahangir,

Thank you for your suggestion. This problem is independent of the treatment. No matter which treatment I give to the cells, this problem occurs. Even when cells remain in culture with regular change of the media without any treatment, cells are becoming non-functional. They still proliferate normally though. Is it because of the high glucose media? Since there are some literature suggest that high glucose may provide some resistance to the cells and it also gives stress to the cells. Normal RPMI 1640 does not have higher amount of glucose as compare to my DMEM media. I have about 22mM glucose in my media.

Thank you,

Dear Prahlad

I will try to help you. I had experience with these cell lines and I had no problem with these cells.

Please write me all steps of your experiments. Is this important to treat the cells for 3 weeks?

Eva

Dear Eva,

Thank you very much for your message. As I have mentioned, I culture these cell lines in DMEM high glucose media rather than most commonly used RPMI 1640. I am looking at the anti-apoptotic roles of different redox proteins in MM. I am using chemical inhibition and RNA interference approach. For me, cells do take one week to reach full expansion stage. In second week, I give different treatment for upto 72 hours and do cell viability assays, caspase assays, and western blotting. Then, if I have to repeat the same experiment in third week, I start seeing different results so I cannot get consistency in my data. After three weeks, cells are still viable but do not respond to any treatment. I am trying to develop drug-resistant cell line and for that I need them to be in culture for 3 months. I have been working with AML cell lines and never had this problem. I also see people using these cell lines and they also do not have any problem and cells are still active after months in culture.

With this problem, it is very hard to get consistent data and have to maintain liquid nitrogen stock and wasting lots of time.

I hope this information will be useful to you in order to help me out. Once again, thank you very much for your help. I was just wondering if I can get your email address, so that it will be much easier to converse with you, if you don't mind.

Thank you Eva,

Regards,

Hi again Prahlad:

I cannot get the reason why you prefer to use DMEM which is (and other media with high glucose content are) mostly recommended for adherent cells (may be due to this adhesion-dependent growth demanding already a high level of oxidative transformations). If I were you I'd switch immediately to RPMI!!!!,.

Hope to hear some update from you!!

Hi Prahlad

I've been using the same myeloma cell lines for a very long time and I've always grown them in RPMI1640  (product number 61870-010 from Life Technologies) with 10% serum. We've never had a problem with them. I would also suggest you check that the cells are what you think they should be. We normally send our cells for STR typing.

Hope this helps.

Regards

Lauren.

Hi Lauren,

Thanks a lot for your suggestion. I have changed to RPMI 1640 media with 10% FCS. My cells are growing much better and I no longer have that problem.

Thank you for your help.

Dear all,

I am now culturing this cell line (RPMI8226) and I had some problems after a week of culture. I observed some adherent cells  that are growing "healthier" than those ones that I observe in suspension (actually they seem to be burst).

I cultured these cell lines in RPMI 1640 medium (Lonza) supplemented with 10% FBS in T25 flasks (Orange Scientific) and the cell viability was very low (30%). I also maintained some cells in 12 multi-well plate (BD Falcon) and reached higher cell viability rates (50-60%).

I cannot understand why they decreased cell viability suddenly (after a week of culture) since they are being cultured in the same manner.

Can you help me with this?

Hi Vanessa,

Are you making sure that your cells are not over-confluent at any point? And not under-confluent either? Also, does your media contain L-Glutamine?

Dear Ema,

I am using RPMI medium from Lonza, which is already supplemented with L-glutamine. And maybe I am keeping cells over-confluent because in multi-well plates I seeded cells to a cell concentration of 1-2 x 10^5 and they are much better now. I am just not sure if cells will keep viable and adapt to T25 flasks.

Thank you.

Best regards,

Vanessa

I find that IMDM+5% FBS + IL6  (we use 0.5ug/ml but this is probably overkill) works pretty well . Also, like Erna said, they like to be cozy, between 5E5 and 1E6/ml works pretty well. After they start expanding you can keep using bigger flasks, I'm routinely using T175 with 50E6 in 50 mls.  I have the feeling that each cell line has its own growth pattern though, so some trial and error will be necessary.

Cheers

Dominique

Prahlad:

What functional assays are you referring to?

I am using F0 myeloma cells with DMEM (10% FBS+1% PS). Should I change the medium?

Hello All,

I have been culturing OPM2 cells with RPMI media +20% Serum. But my viability is not reaching up beyond 70% and the cells burst or shrink soon. Has any one worked with OPM2 cells and faced similar issues.? I understand that these are slow growing ones, but for me these cells dont grow at all . . . !