I agree with Pedro, I would pull another isolate from the freezer or start with a fresh isolate. Sorry for the bad news.

I agree with Pedro nevertheless test for the presence of mycoplasma also.

Thanks very much for your help! I thought this may be the case!

What supplements are you using for MSC culture? If it is FBS, switch to human platelets lysate, and they will grow much faster, and morphology is much better. This is the only/best solution. Please contact me if I you need any (extra) help.

What is the media formula and where do you buy human platelets lysate?

I also agree with Pedro and other scientists. When culturing must comply with many conditions. It is important to choose supplements, for example the percentage of FBS. Check the possibility of contamination. To sum up, we must strictly observe protocol of experiments.

I also agree that these cells look senescent. I would recommend to thaw a new batch and prepare fresh media supplemented with FBS from a lot that you know is good. Culture medium supplemented with platelet lysates might affect several parameters of the MSC and thus you should be careful.

It happened to me, when I was growing the cells with DMEM 10%FBS . The same cultures with the flask coated and with serum free medium were growing much better, even if I tried to increase the FBS percentage it was not working. My advise is try with another medium or try to coat the flask with fibronectin.

Because we sometimes forget our scientific distraction simple rules. We must comply with all stages of approved research protocols.

Yes, the morphology suggest senescence. Too bad that this already happened at p6, but not too uncommon either. Good luck!

HI I do agree with the rest of the group. From your photo they do look as they entered senescence. In our lab we normally use them for phenotypic/differentiation studies and rna/protein work at as early passage possible. We go sometimes up to passage 4 or 5 the max if we are desperate as it is not uncommon they will start senescing soon after that. I would rather chose another vial to start than changing your medium as if you want to compare you results with previous work it wont be directly comparable. Good luck

Dear all, thanks again for the replies. We retrieve the cells initially into DMEM w/ 10% FBS, 1% pen/strep and glutamine, then subculture some days later into Mesen pro (Gibco) which is a reduced serum medium for MSCs.

- Check the age of the donor. Older is the donor, lower is the growth of the cells and faster cells reach senescence. Consequently some MSC cultures reach senescence very rapidly and to not grow very well.

- human MSC reach senescence quite rapidly. It depands on the number of population doubling. P6 (PD?) + previous passages in a other lab seems however to be already a lot. Try to limit the number of passages and to optimise cell amplification by replating the cells at a low cell density.

- Platelet lysate is also a very good additive for human MSC and gives very good results in terms of growth of MSC when compared FBS qualified MSC . We are launching a standartized human platelet lysate on the market ; We did a lot of experiments and works efficiently.

- Addition of bFGF 1ng/ml can also improve significantly the growth of your MSC.

Plasma contamination or that maybe the nitrogen dose caused to be slowly growth of MSCs ...

From your image, I'd agree that this is not a normal morphology for MSC and that these cells are probably senescent. This could be expected as these cells are now at p6. We usually use our MSC (obtained from young, healthy donor's) up to p5.

When my MSC are growing slowly sometimes I find that passaging them 1:1 and giving them 50% media changes 1-2x per week can help their growth. I think the reduced media change allows secreted growth factors to build up in the media and promote proliferation. Hope this helps, Good luck

As Michael Rotherham has said. during my experience with hMSC i woul never use post passage 4.

We also prefer to reduce post passage.

Dear Jonjo,

Usually human Bone Marrow MSC seize to proliferate at passage 5 but in my hands Adipo and Umbilical Cord MSC can be passaged for longer periods. Also the initial plating density should in my opinion be 500 cells/cm2 and the medium should contain 20% FBS to help them grow after freezing. But nowadays most people seem to look at the population doublings the cells have undergone in order to check their potential, so if you can figure them out and of course know the source that will help you a lot.

I hope this was helpfull.

I completely agree with olga. Bone marrow MSCs slow down their growth by the end of Passage 5. And you are also shifting the cells from serum containing condition to serum free condition. That could also be one of the reason for their lower proliferation.

From the cell picture, I think it is better to discard them and ask for new MSCs ( early passage and younger donor). You can try to use Loza MSC growth medium(serum free medium) if you do not mind to waste your time.

My thinking you can check a concentration of FBS. Usually we use 10 - 15% FBS, it is expensive method but we can keep on normal MSCs

By looking at your cells, it is clear that they are very old and undergoing senescence. I suggest not to use these cells anymore. Usually, healthy MSCs are small and look spindle shaped. The higher the passage, the bigger they become. Also MesenPro medium supports good MSC growth if you want to try with your next set of MSCs.

Hi, I'm currently studying senescence of BM-MSC. I agree with others saying that your cells looks senescent and you would better discard them and use some at a lower passage.

Anyway, in my lab experience p6 is a quite low passage to have cells that completely stop to proliferate. In our culture conditions (alfaMEM+20%FBS, seeding 2000 cells/cm2) they reach p10-p12, sometimes more, before they become senescent.

I suggest you, before starting all over, to consider all the factors that may have influenced your culture (e.g):

-age of the donor

-pharmacological treatments of the donor before (es. chemotherapy, prolonged cortisone)

- quality of the BM withdrawal (we think this aspect is underestimated)

-freezing/thawing procedure

-culture condition (also a different lot of serum can impact, besides we found that seeding density the first split is not so relevant)

In the best culture condition, the donor variability is still the main factor, so my advice is to maintain the record of cell count at every passage to create a proliferation curve and to calculate Doubling Time and population doublings. In this way you can check if you have a good donor.

Our best cells maintained a doubling time < 3 days (that means healthy cells) until p9.

Hi, your cells look senescent. Usually MSCs at a lower passage are more fibroblast looking(spindle shaped). As the passage number increases, the cell size also increases (nuclear to cytoplasm ratio) as these cells tend to reach senescence pretty early. In my experience 10-15% FBS should be sufficient to maintain them in culture. Please check the lot # of the FBS you are using. Also like the other researchers have mentioned, the donor's age, sex, disease state, pharmacological history (eg, steroid therapy) etc should tell you something about the cells.

Also, when you change the media, just change 50% of the media.

Try to use lower passages for your experiments (p3)

I hope I answered to your question

All suggestions may be right, but you may try to revive the culture.

Do it by using low glucose DMEM +1% glut (avoiding addition of non essensial aminoacids) +20% (!) FCS (Gibco, usually works well for bone marrow MSC). The senescent cells may die, but the plate will be taken by "young phenotype" cells, if some are still present in your culture.

Senescence can be detected by a senescence-associated β-galactosidase assay. I have used MSCs that were cultured after immuno-depletion with MACs after isolation from BM, to have a more homogenous population at earlier passages, and I found I could passage them at least to p.10 without becoming senescent, as well as retaining essential MSC markers.

you can add serum in the media and check if their is any increase in growth and proliferation. In my opinion, p6 is quite an early stage for cells to become senescent.

The cells look senescent. Unless you have done two things (1) used a specific lot of serum that has been pretested for its ability to proliferate MSCs and (2) that you included FGF-2 in the medium, human MSCs will senesce in 3-5 pasages if they actually grow at all. Even with the optimal serum lot and FGF-2 included, be very wary of extended monolayer culture - MSCs do not retain differentiation abilities forever. Minimize population doublings for best results. Also, be aware that companies claiming their serum is good for MSCs are not necessarily right. We roiutinely have to test up to 10 lots of serum to find one that proliferates the cells very well while retaining their differentiation capabilities. I recommend you start with fresh cells.

MSCs and all human and mouse cells grow better at lower oxygen level. You are probably growing them in 21% atmospheric oxygen and you should try 2-5%. I also think you should get a younger passage and keep the volume of your medium high (need to be sure you are not concentrating salts in your medium by evaporation). Hope this helps 

I agree with Deepti Singh: check always your cultures for mycoplasma (see Marx, Nature Meth.  May 2014)