Hello,

those cells look senescent.. This can have some reasons:

1. You got old cells (with high passage number)

2. You seed them in a too low concentration, respectively a too low number of cells survived the process of thawing. This means the cells need several divisions to fill the culture flask. A high number of cell divisions accelerate the ageing og the cells.

Did you have a lot of cells that did not adhere 24h after seeding ? If yes, this indicates that a lot of your MSC did not survive the thawing and seeding procedure.

Best regards

Dirk

The cells are not looking healthy. They have become apoptotic. Since they were healthy to start with something might have gone wrong during the culturing of these cells. The cell numbers that you seed is a very important factor for the cells to grow. I have been working with the MSCs for almost a decade now. This behavior of cells are seen when the cells seeded in the correct numbers. I seed 0.6 million cells in a T-75cm2 flasks. Also i would request you to check the incubator conditions such as the temp, CO2 and humidity. You can start a new expt if you have cells and ensure proper number of cells and i guess your media would be fine since you could passage them for one passage. With the present cells the even if you trypsinize them and replate it will not help since they will not grow their viability will be very low. You can revert back if you any further clarifications or help

I agree to the suggestions of Dirk and Pedro. Seeding of cells in low number also results in slow proliferation and growth rate and facilitates senescence. One more possibility which might be the cause is

> if you are using high concentration of trypsin or exposing the cells for longer time to trypsin will also cause cellular injury and reduction in the number of cells attached to the surface.

Hi,

I do agree with previous respondents. I had the same situation some time ago, and got in contact with Lonza (where we bought the cells from) to check with them weather they had any complains with that batch of cells. We were using alphaMEM formulation as well, but we were changing the media every 2 days. That was detrimental for the cells, since we were not allowing them to interact through their secretome. So, the advised medium change time frame is 3-4 days.

Hello!

When growing MSC start becoming "round" in shape this is most often an indication that they die. In the case that they detach this is for sure death. If death comes as a result of contamination (which does not "show "clearly in the MSC cultures) or senescence needs further examination. The truth is though that cells that become old have again a spindle shape but are quite wider and also have "dots" on their surface as one looks at them under the microscope.

Good luck !

H.D

Dear Chi Woo Yoon,

So you probably figured your cells are differentiating. in the future, add 5 ng/ml of Basic FGF to the growth media evey other day.

Good luck:)

Usually hMSCs keep their stemness characteristic to 6 or even more passage. If the cells were ok during first passage, probably there are some problems in your sub culturing procedure.

It is hard to see well the morphology of the cells from your picture but MSC' morphology always change with further passages, quicker for cells from older donors longer for the cells from pediatric donors and depending on the numer of doublings they did during a passage. Additionally lack of or addition of growth factors may influence how long you will have them proliferating but finnaly all will get more elongated and will decrease proliferation rate.

In my cultures I always start from bone marrow sample, isolate MSC enriched population (CD271+) and start culture. I spilt them with accutase 1000 cells/cm2 after CFU-F colonies grow. They look nice, especially from pediatric samples, adults have lower CFU-F numer in CD271+ population but grow nicely as well. I add growth factors to culture medium.

Hi there,

If everything is ok with your media, incubator, and harvesting/seeding methods, and if you followed the instructions exactly as depicted on the datasheet for these cells, I'd just contact Life Tech and get them replaced by cells from another lot. Faster and easier. :)

Dear Chi,

your cells are senescent. MSCs espcially from bone marrow are known to senesce fast. Senescent cells are known to be flat, enlarged and have limited proloferation and differentiation capacity. Try MSCs from a young donor, or change the location of MSC (e.g. adipose, umbilical cord or hair follicle). These MSCs are known to proliferate faster and senesce slower. You can also supplement your media with bfgf. Plate your cells at a density of 3000/cm^2.

Good luck,

Panagiotis

Thanks all for answers.

For seeding density, I used 5000 cells/cm2 which was suggested on instructions

and I didn't see too many cells floating (actually most of cells were attached), So I guess it was not density issue.

ِDear chi woo yoon

Please extract the RNA and survey the mutation.

Good Lock

Ahad

I'd suggest checking the instruction - sometimes when you buy primary cells which are not directly from isolation you can find a note with limitted lifespan - in my case it was only 1 passage left. Maybe a change of cell source will help in the future

dear Chi Woo Yoon, i suppose (as most of previous scientists did) that these cells are either senescent or "sick" a little. however such morphology can be the sign of some differentiation processes, so you should use any available test to check this probability. also it's really important to know individual features of cells' donor 'cause it can affect the quality of culture a lot. as you've mentioned the seeding density 5000 cells/cm2 you can hope that it's really not the density issue, but anyway it's always better to check it again using different variants of density. moreover, i've noticed that one of the main features of mscs is their "flightiness". any changes in co2/o2 ratio, trypsin, flasks, growth media or serum can alternate their morphology. so maybe the cells just don't like the current place :)) anyway - do maximum of available in your lab tests and share the results with us here :)

Hello Chi,

As per my experience to hMSCs, I found that they change their shapes after about 10 passages but not at 2nd passage. Are you feeding them twice a week. They do become flatten and enlarged with some pseudopodia LIKE STRUCTURE and grow very slow. They remain in culture live for long time. The passage of these cells differ from cell to cell isolated from specific tissue. PLEASE ADD SOME GROWTH FACTORS IN THE MEDIA TO MAINTAIN THEM IN CULTURE WITH PROPER SPINDLE SHAPED MORPHOLOGY FOR PROPER MULTIPLICATION & GROWTH.

ALL THE BEST !

Dear Chi woo Yoon,

I completely agree with Dirk Henrich's suggestion. I would like to add one more point here is, even we see sample to sample variations. hMSCs isolated from some samples continue growing till late passages like p15-16 with constant PDT however few reach senescence after P4-5 in spite of maintaining high seeding densities same culture conditions with growth factors. so me reason is still unknown.

Dear Chi,

MSCs tend to undergo senescence at higher passage numbers for sure but sometimes you could find MSCs from a few donors which tend to undergo senescence even in early passages.Such MSCs with low proliferation capacity could function differently from the highly proliferating ones.,for example higher paracrine secretion. MSCs do exhibit differential proliferation capacity from varied donors and it still remains a challenge to identify the reasons behind it.

Try using a mitogen for your cultures,maybe FGF-2 could be a good bet.

I suggest you check your media for mycoplasma too and also the media composition and the lot of FBS.

Good luck

Dear Chi,

Your MSCs were undergoing senescence at passage 2. It looks just as you said "cells were bad from the start". Since there is big biological variation between MSCs from individual donors, I suggest to select good cells for culture and experiments. On the basis of good cell source, you can increase MSC growth rate by adding mitogenic growth factors etc.

Dear Chi, did you check for mycoplasma contamination?

Dear Chi,

The main reason which may cause change in morphology and stop or slow proliferation is senescence. It means you should be sure of Mesenchymal stem cells to be of low passage (3 or 4 is ideal). You can test their senescence by Lactate dehydrogenase test.

An other reason could be their differentiation initiation which may originate from supplementing the medium with more than 10% inactivated FBS.

Of note that some times change in optimum PH of medium or CO2 concentration within the incubator lead to slow proliferation.

Hi Chi,

I have experience with primary hMSC. I've checked similar cell morphology in cultures with high passage, around passage 9 to 10, when the cells were cultured around 10^4 cells on T25 flasks in each passage (considered low concentration). They grow very slowly, taking around amost 2 weeks to get confluent on these passages.

I don't know if you observed this growth pattern on your cells. In my cultures they seem to present a slow metabolism too, because I could not observe changes of medium colour.

Just to discard the hypotesis of mycoplasm contamination i recommend you to use some mycoplasm detection kit. Because these kind of contamination also can retard cell proliferation and make them senecent. It's very difficult to see mycoplasm only by microscope as we see fungus ( white spots like cottom) or some bacteria ( the medium present a milk colour and we can check by microscope some black spots of bacteria colonies shaking). So people usually uses some PCR kits.

Another important thing is to change the medium every day or every 2 days because during early passages the cell metabolism is higher. They get confluence around day3 to 5. And Avoid let them hit 100% confluence. This also may induce cell differentiation.

The use of FGF2 or FGFb or others growth factors are reccomended as supplement, but some works showed it can interfere in differentiation induction experiments (Gharibi& Hughes, 2012; Hagmann et al., 2013). So, before you use growth factors make sure what are you going to do in future with these culture.

One very well studied MSC marker used to isolate this cells is Stro-1. After passage one you can sort Stro-1+ cells by MACS (magnetic sorting) or Flow cytometry. And after that continue your experiments with a enriched Mesenchymal Stem Cell population. This can be an alternative to save your cells, if you discarded all problems before. Without sorting you have to consider your cells as mesenchymal stromal cells, according to International Society for Cellular Therapy ( Horwitz et al., 2005; Dominici et al., 2006).

Cell culture really is not easy to work with, but worth a try. Good luck

I hope it help you

Hi,

We are growing MSCs and experiencing some difficulty, our cells seem to barely divide (the number we get out via counting with haemocytometer seems to be the same almost as that we seed some days previously). However they do seem to grow slightly and become more confluent. I was wondering if it's the case that our cells are senescent? Can anyone advise?

Jonjo Miller,

What is origin of this MSCs and at what passage?

As you correctly mentioned even I feel they have reached senescence also they look unhealthy. Still you can take advise from experts.

Thanks

Akshata

Hi, I agree with the comments given above: your cells look senescent. As you said that you had just bought them from Life Technologies and this seems to be your second passage, this effect should not have happened that early in the culture. The reasons for this might be that the cells were already at a high passage number when they were shipped to you (which I do not believe), or damaged during transport or - the most likely explanation - did not react well to your culture system (e.g. culture plastic, medium, supplement and seeding density). This is of course a tricky issue, but you could try to include different types os cell culture plastic ware and media for your culture if you are willing to try again. Good luck!

Dear  Chi, 

I would suggest a kit to evaluate cell senescence. Anyway, I think you are using a too poor culture medium. You can try to use DMEM/F-12 with 10%FBS and 2 ng/ml b-FGF. Please, find attached a paper. I hope you may have further suggestions.

Best regards,

Luigi. 

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