I recently acquired MCF10A cells and also find them to be very fast growing. I use the same growth medium as you do. I think you should consider the possibility that the fast growing phenotype is the appropriate one, since your cells were newly purchased from ATCC. The authentication procedures used at ATCC are known to be very reliable.

How many passages have you performed to the cells before they start growing that fast? Maybe they mutated.

Dear Michael,

I recommend to check the identity of your cultures, since slow cultures are very often a victim of aggressively proliferating contaminating cell lines, e. g. HeLa. The number of false cell lines in circulation is unacceptably high (15 to 30 %) and the use of cancer cell line models is impaired by reliance on misidentified examples representing entities with biological characteristics different from those supposed.

Best regards,

W. Dirks

My first question would have also been about passage number, as above. The second question, have you changed the brand of media? Not all DMEMs are created equally, some have much higher glucose. Another possibility is that the termostat in your incubator is off and the cells are growing faster in response to a slightly elevated temperature. Also check the CO2 regulator.

I agree. When faced with a sudden and dramatic change of growth patterns, I would first try to rule out contamination by a fast-growing cell line.

Dear Michael,

I agree with Wilhelm Dirks, contamination with other cell line is possible and plausible.

If not contaminated, did the cell line mutate or did you change the batch of horse serum.

In earlier days, I experienced the growth of MCF-7 cells could change dramatically with different bovine serum batches. Normally, estradiol receptors do not play a role in the growth in MCF-10A cells, but if ER is involved again, estrogens can improve the cell growth. In this respect you can treat the serum in advance of your culturing with activated charcoal and/or DTT (dithiothreitol). Charcoal will adsorb the small molecules, like steroids and estrogens, while DTT will inactivate the proteins with SH groups, like insuline EGF, IGF-I.

Best regards

Willem Schoonen

MCF10A is a breast imortalized cell line with very fast growth rate. In our lab, we use HuMEC medium formula and passages are at 2-3 days after a subcultivation ratio or 1:6 to 1:10. That was always at same for several years. Is it possible that at first time your growth rate was too slow?

are you keeping other cell lines which grow very fast? maybe you can perform some characterization to confirm they are still MCF10A.

Are you using serum free medium. After a while, serum sort of "transforms" (don't take this literal here).

In my hands MCF10A cells should grow very rapidly, similar in speed to HeLa or HEK293 cells, with doubling times under 24 hours. In addition, MCF10A cells spread out very nicely, so they can give an appearance of very fast growth. If you have ruled out contamination by a different cell line, then my suspicion is that previously your MCF10A cells were growing slowly before (maybe poor batch of serum, media, or some other component) and they are now growing at their expected rate.

To illustrate just how fast they are growing, I have on occasions split MCF10A at 1:60 and they still fill up a dish in less than a week. (Not recommended if you need to use the cells for important experiments.)

I agree with others that the most likely answer is that you have contamination with another cell line. This occurs frighteningly often in many labs and it is therefore always advisable to regularly validate the identity of all the lines you work with.

throw them away. They r not good.. they have lost their identity. Get a new vial from ATCC

That is how these cells are. There is nothing unusual about that. Though they are normal breast cells, their growth rate is such. If you want to use a slower growing normal cells, you may want to use MCF-12A. As long as you do not have doubt about the cells being contaminated, you should not worry about the cells.

As many other investigators expressed the concern that it might reflect contamination with some other rapidly growing cell line. Moreover, MCF10A and MCF12A lines need very stringent culture conditions. They respond to stress (e.g., change in CO2, medium, overgrowth etc) rapidly by turning on some stress response genes (PLoS One, 5:e13390,2010) which may attribute to their altered growth pattern.

It is OK and NORMAL. One the get over confluenent for first time after refreeze they start growing this way. Nothing to worry about. Just split them over the weekend 1:15 and 1:10 for 48h

I split mcf10A twice a week at the ratio of 1:10 .I think that they are growing too fast but the morphology is o.k

Hi! For how many passages have you grown your cells? It might be that they have changed because they are old.

If the problem is cross contamination with another cell line (with the same morphology and very high growth rate, more than MCF10A growth rate) the solution is easy: Thaw a new frozen criotube. Is an easy step to resolve this doubt, but sincerely I don't bealive that there is the problem.

I would recommend that you get rid of the EGF from your medium and see if you can reduce the cell growth proliferation.

As far as I know, cholera toxin increase cell growth for MCF10A.

You can read more from Cholera toxin: A paradigm of a multifunctional protein, Indian J Med Res 133, February 2011, pp 179-187. They divide approximately every 40 hours for me.

Check Mycoplasma contamination.

two different cell lines at the same time responded does eliminate the possibility they are mutated. Since the growh advantage is a function of selective pressure, this is very much unlikely happens at the same time in two different cultures, hence they are different cell lines. However, I agree with Dr. Mehta, the stress induction might cause this kind of response in cell culture. I suggest checking mycoplasm contamination first and change the CO2 supply. To slow down the cultures you may consider using DMEM instead of DMEM/F12 mix. good luck.

Probable intracellular mycoplasma infection. See this link as an example: http://www.ncbi.nlm.nih.gov/pubmed/16054780 "when infected by Mycoplasma fermentans, human B lymphoma cell proliferation increased strongly". There is an entire industry dedicated to the eliminating of mycoplasma species from research cell lines.

I have never had very fast growing MCF10a cells! I would suggest that they are contaminated with a faster growing cell line. If my cells suddenly started growing much more rapidly I would chuck them and thaw out fresh. You cannot really trust the data and make any realistic comparisons with previous experiments.

If it is mycoplasma and both lines are contaminated, and both respond by accelerated growth rates then the infection is rampant, it has probably spread and colleagues with other cell lines are probably experiencing (and posting here maybe) about slow growth rates.

Kemal makes a good point regarding mutation.

Have you changed any of your reagents-different serum batch, or made up fresh stocks of cholera toxin Pred, EGF etc? because it might be that you are seeing the effects of increased mitogenicity of your additives. I would thaw fresh cells and make up fresh additives.

You should check how many passage you did. Usually, old cells change their phenotype.

Hi Michael,

first of all, thaw a new vial of cells. If not possible, reduce EGF and cholera toxin 10 ng/mL and 50 ng/mL. Try it.

Good luck

Marcelo's suggestion is good, it could be that your EGF and CT is of such good quality that whilst you are adding what you are advised to add by everyone, in terms of concentration, you may be adding more as the specific activity is higher. Maybe try a few different concentrations of both till the cells behave how they used to. Good luck!

I recently acquired MCF10A cells and also find them to be very fast growing. I use the same growth medium as you do. I think you should consider the possibility that the fast growing phenotype is the appropriate one, since your cells were newly purchased from ATCC. The authentication procedures used at ATCC are known to be very reliable.

Possibility 1: Lynn is right.

Possiblity 2: Your cells have been transformed in your culture. My MCF10A cells grow extremely slow, but at the same time, I don't know how "OLD" is the frozen cells. I would expect normal cells proliferate rather slower than cancer cells.

In strong agreement with Lynn Thomas! In my experience with these cell lines they grow too slowly if they are in the wrong media- probably what you have thought was "normal . In the correct media as suggested by ATTC, including cholera toxin you should see a faster , but normal growth. DId you change supplement sources? THe ones from the kits are your best bets

You should try remove the toxin. I have MCF-10A without this componente and they are growing well. However, you should consider that this cells is changing its phenotype, mainly if you are doing many passage.

Good luck

Marcelo

I believe she said she was working with early passage that was frozen early. Anthony and all, remember that while these are not cancer cells , they are transformed cells which may grow just as fast as cancer cells. Transformed cells are at least one step towards cancer. The chemical used for the transformation is of course a mutagen, hence the transformation. These cellsare likely be cancerous when returned to an in vivo situation

Hi Michael,

My MCF10A also grows very fast. But the speed has been very steady. It is cultured in 10 cm dish with complete media ( almost the same as your recipe, but with antibiotics). I normally begin with 20% confluence, and they surely will reach 100% at 48h and need to be passaged.

I once did a small test to culture MCF10A with Fetal bovine serum and without cholera toxin ( other components are the same). Cells were fresh thawed(2 months storage), which eliminated the influence of previous complete media. They worked well. They grew a little bit slower , but not significant. ( 72h, surely 100% confluence and required passage).

In my case, some cells frozen for a while ( >=half a year) sometimes require several passages to recover. When they are happy again, they grow fast. (Especially if temperature turbulence took place while stored).

If you afraid that your cells are contaminated with other kinds of cells, you can always extract DNA and do STR to check. It's fast and cheap.

It's safe to check mycoplasma, especially if your experimental model is very sensitive...

After all, I am happy that they grow fast. I don't have to waste my whole lifetime waiting ;D.

Best,

JF

My bad, I was thinking of the chemically transformed 184b5 companion cells. We are working with these alongside the MCF cells.

My MCF 10 A cells are growing very slow. I really wonder how to boost their growth. Plz suggest!

Check passage number. When cells become senescent they can grow slower, faster or change their morphology. I think the problem could be the cells and not the culture media. Not discard the possibility of cell line cross-contamination.

What were the results from the growth in complete media with FBS in stead of HBS (horse serum)? Curious.

I use DMEM as medium culture, jusr 3,5g glucose and 1,5 NaHCO3 and the growth is normal, not too slow, neither too fast.

Dears Researchers, i am starting a new culture of MCF10A purchased from ATCC. i prepared the complete growth medium as follows MCF10A cells were cultured in DMEM/Ham's F-12 (GIBCO-Invitrogen, Carlsbad, CA) supplemented with , 20 ng/ml epidermal growth factor (EGF), 0.01 mg/ml insulin, 500 ng/ml hydrocortisone, and 5% horse serum. i could not purchased cholera toxin in anyway here in DOHA so it was not added ( but others suggested that this might be ok)??? the problem s my cells are growing slow. it needs around 9 days to reach confluent. when i passed the cells i only counted 4 million cells total / T75 flask!! is this usual???? i think it is way low although the viability of my cells were 95% when counted by trypan blue under microscope. Can you please suggest any modifications to fasten my cells growth??? note: to harvest my cells i use trypsin 0.05%, EDTA for 20 minutes and stop by trypsin inhibitor 1x.

Growing slowly is normal. But the more you grow them, the faster they will grow.

Thank you dear Lui. How many mcf10A you can approximately have in almost confluent T75cm flask???