I feel like I'm starting to bash my head against a brick wall, so if anyone can shed some light on the problems we are having in our lab I'd be most grateful!

We are culturing cloned equine dermal fibroblasts. When they were originally established, they were proliferating really well, up to about passage 17 with nice regular morphology. However, last November we ran out of the original batch of fetal bovine serum, and ever since they just haven't been growing properly - clumping, not forming monolayers, just plain not growing at all, with highly irregular morphology, and they dont appear to be migrating across the plate as well as they used to.

We have batch tested multiple batches of FCS, and twice now we have had a batch that appeared to improve the growth, so we bought a lot of it, but then about a month or 2 later, the same problems with the cell growth have reoccurred. I am currently also testing some plant extracts for their effect on the cells, and some of these extracts are stimulating proliferation of the cells to a very high level. These cultures still have 10% FCS in, plus the extract. So the only difference betweeen them and my controls is the presence of the plant extract - which strongly suggests that the FCS is lacking some nutrient that the cells require.

we've checked everything else that we can think of - media/trypsin/other solutions used on the cells/the incubators have been checked (though we are thinking about buying some new filters for them). the cultures definitely aren't contaminated in any way. is it usual for cells to be this sensitive to FCS batch? My supervisor has never seen anything like this before and is as confused by it as I am. I currently only have about 4 months to finish the lab work needed for my PhD and this is seriously holding me up now.

any thoughts/suggestions really really needed!
Many thanks
Emma

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If you are dealing with primary cells, they have the property to go to senescense (must check spelling:-) after some passages. Beyond that, they either get immortalized, or don't grow whatever, so your primary problem may be primary, not FBS. In that case, make other primary and do your expts before passage 20.

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Thanks for the reply cellcounter

Unfortunately I dont think it is the fact that they have entered senescence because they will still grow when stimulated with other things. (Unless the lack of something/presence of something in the FCS is prematurely stimulating them to senescence). In the past they have certainly gone far beyond the passage number that I'm currently working at before thay enter replicative senescence. I have been highly tempted to restart them from scratch with new primary cells and clone them up again, but to get them to the point that i can experiment on them takes 2-3 months, time I just dont have anymore.

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Two questions:

Have you mycoplasma tested the cells?

What source is your FCS/FBS?

Do you Fyrite your CO2 Incubator(s)

Regards

Rhombus

Both very important, I will be interested in your replies.

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Hi Rhombus,

Yes we have tested for mycoplasma and that came up negative. The FBS is from invitrogen and is heat inactivated from south america

and seeing as i've just had to go google fyrite, the answer to that one is no! but I shall go look into that further if you think it might be a sensible protocol.

I should note that I've got two cell lines - one oral fibroblasts and one limb fibroblasts, and the limb ones are growing perfectly in the same media that the oral ones wont grow in (but originally did before the fcs batch change)

thanks for you help

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Well done for your answer to the FCS...very comprehensive........you are fighting a losing battle however!!!!!!
We do not heat inactivate any more...this used to be done to inactivate compliment, but with ultrafiltration these days, it is no longer required or recommended. Also South American serum is one of the poorest quality serums you can by. So by heat inactivating you are further reducing the quality of the serum by denaturing vital ptoteins required for cell growth.

Mycoplasma testing... again very good by having a negative test....however which test did you use????????...again very important.

Cells from different regions of the body, albeit fibroblastic, will grow at different rates.

Fyrite testing is essential to maintain precise and accurate CO2 levels over months/years of subculturing. Also very important to keep the incubator humidied.

Again kindest regards,

P.S. Dom always complains I am too abrupt. I am soft and cuddly really.

Rhombus

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