Is it possible that your cells are overgrowing? Maybe the media (high glucose) is causing them to proliferate faster and they are now growing to confluent? Also, sometimes the type of transfection reagent (lipofectamine, effectene, etc) can also influence the viability of the cells after the transfection.

Something else, which might sound a little silly, but what are you actually transfecting the cells with? what do you expect to happen to the cells when you add your gene?

Dear Sandra,

did you try to culture just the feeders with the iPSC medium (without any transfected cells on top)? We had a similar problem during the selection of ES cells. After 2 days all the feeders died and thereby very efficiently removed the ES cells from your culture.... Finally we realized that our feeders were not able to survive in the selection medium. We prepared fresh feeders and the problem was solved.

Best

Volker

Dear Sandra,

Which type of coating are you using? If cells are overgrowing a polybrene coating could help.

Usually my when my cells do this it is a problem with either the media or CO2 levels. Check CO2 levels and re-calibrate if neccessary. I concur with Volker, try fresh feeders. Can also try to change media more frequently.

Sandra,

I agree with Leornardo, check you coating. Also, are you using irradiated feeder cells? If so, cells should not grow. I see that effect when my prep of irradiated feeder cells is not great ( too much irradiation). Before starting the reprogramming, make sure that you feeder cells are in good condition.

Good luck

My experience is with respect to BM-derived stromal cell lines used to support hematopoietic differentiation. For these cells it is important that they be maintained at a relatively low passage and/or that their time in culture be short–once a vial is thawed we never use beyond 2 months before thawing another one. For us, in cases where lifting occurs it has been because the cells have been in culture too long ~ 2 months or more.

Dear Sandra,

If you are not facing a general cell culture problem (media composition, CO2 level etc.) you might try to coat your plates with matrigel. It helps to maintain your cells attached and supports your reprogramming – usually you do not need feeders at all when using this coating.

You could also wait a little longer before switching to the iPS medium. Usually iPS media contain no/ little serum or only serum replacement and most feeders do not like this.

One last thing, do you use antibiotics while transfecting your cells? Sometimes this results in low viability.

Best

Marc

I meant poly-l-lysine coating, sorry.

Check the composition of media. MEF do not grow well when using for example KSR instead BFS. I do not think overgrowing is the problem I agree with Volker Eulenburg. The media have to be the problem

please confirm if the coating before seeding feeder cells is changed. For example, if you use Gelatin type A, instead of type B which you normally use for irradiated iMEF cells, then you will find iMEF leave apparently 2 or 3 days later. Also the viability of feeder cells in the medium they are not supposed to face needs to be tested. About density issue, I do not really it is the main reason either.

are you doing research on murine reprogramming ? If you studied murine somatic reprogramming. It is not necessary to use feeder in somatic reprogramming. In my lab, we do not use feeder , only if we culture completely reprogrammed induced pluripotent stem cell. what is your ips medium ? Is it for mES culture ?Maybe there is something wrong with your medium .

Which methold do you choose to treat your MEF, mitomycin C or radiation? If mitomycin C, you can optimize working concentration.

Hi everyone, thank you very much for your ideas =), I really appreciate them. Yes, my research is in murine somatic.

I work with MEFs inactivated with mytomicin-C (3h inactivation). I use passages

Try to culture feeders only in your iPSC media, and in media with bovine fetal serum instead KSR

We usually split our cells between 3 to 5 days post-infection/ transfection.

KSR is fine for reprogramming and -in my hands- better than using FCS-only as serum supplement, but if your feeders do not tolerate SR you could add 1-2% FCS to your medium without loosing much of KSR's beneficial effect, while helping your feeder cells to survive.

Have you tried to cultivate your feeders and iPS cells with the iPS-medium only? Do your cells tolerate the medium under this condition? Once we had a problem with our ß-Mercapto stock - it is rather toxic when used at too high concentrations.

If you still face problems with you feeders I would also recommend to reprogramm without feeder cells. For mouse cells reprogramming works also fine without them - although you might get less total numbers.

If efficiency is a problem you could still add small molecules like Chir, SB, VPA etc.

I haven't checked how do feeders respond to iPS media, that should be a good start. I will check that and after that "correct" the FCS serum concentration in the medium. Marc, when you say "split our cells" that means placing them on feeders?

We haven't tried to culture the cells without feeders, because medium supplements needed are way more expensive than feeders, so we are looking for "cheap" alternatives to solve the problem due the budget is limited.

Thanks

Hi Sandra, yes that's what I mean. We place our to-be-iPS onto feeders after 3-5 days. Nevertheless, it should also work without this step. Feeders are more important when expanding iPS-lines.

Maybe you try a simple protocol (without re-plating) first and optimize if it does not do the job.

Thanks Marc.

I just did a transfection yesterday and I will prove growing them without passing them onto feeders, until I "see differences" between my transfected cells and controls. I am planning on growing some plates with "normal medium" (DMEM-F12+10%FBS) and some with IPS medium, to see if there is any difference in develop, nevertheless I think that the ones that will grow in IPS medium would start to detach.

Any recommendations?

Hi Sandra, I think you are on a good way. Now see what happens. If your cells die in both conditions (DMEM-F12+10%FBS and iPSC-medium) while growing fine without transfection you probably face a toxicity problem related to your tranfection.

If your cells tolerate the 10% FBS condition but do not like the iPSC-medium you will know that the latter is to be blamed. As mentioned, I would also try to grow iPSC-lines or ESCs with your iPS-medium – If they also do not tolerate it it’s again the medium.

If growing iPSCs works fine and transfection is also no problem you could try to find a compromise between iPSCs and somatic/ feeder cells by mixing your reprogramming medium with 2% FBS at the beginning (for example the following medium works fine in my hands: DMEM/F12, 2% FCS, 10% Serum Replacement, 100 uM b-mercaptoethanol, 2 mM L-glutamine, 1x NEAA and Lif)

One last thing, it might sound trivial but have a look at your media bottles, some media have to be supplemented with glutamine or pyruvate (usually marked by a „minus“). Without adding these components your cells die after a couple of days.

Hello Marc, thanks for your advice I would check all the things that you are pointing out. Sadly at my lab we do not have other iPSC or ESC lines, so the iPS medium proof can not be done. I just changed my cells media today, I will be hoping for the best and notify you any happenings.

Thank you very much, =)

Thanks Frank :) I have done those tests :) My feeders are working good already, I had a problem in the inactivation process, but it is already fixed.

Thank you all!!

I was about to say it was a problem of residual mitomycin, or with not efficiently inactivated feeders.... anyway, after addition of reprogramming factors, we always seed the cells confluent on their own medium... at days 3 0r 4 we perform a split and that split is divided much diluted onto feeder cell layers... the efficiency of transfection is another crucial parameter.. it seems that viral transduction is much more efficient than transfection methods... then it would be 'normal' that all majority of not transfected cells would detach...

Try decreasing the concentration of FGF. iPSC need it but MEFs don't like it.

have you checked the effect of antibiotics that you use for selecting iPSCs on feeder cells?

I have found that KO 'knockout' DMEM from Gibco works better than F12. Not sure why though! The feeders seem to like it a bit more.