3T3-L1 cells die in culture

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Aspeedasai
Aspeedasai's picture
3T3-L1 cells die in culture

During differentiation my cells start detaching from the culture dishes. When fresh stocks are thawed and plated die. I am suspecting contamination with mycoplasma. Is my doubt valid? Could there be a possible contamination in my culture? I am going to have them tested for a possible contamination, but if anyone has experienced similar problems please provide your comments.

Very frustrating as one has to wait for a week to complete the differentiation. While when cells die, each time you thaw them amounts for months of wasted eforts. Has anyone has had similar problems? if yes, what solutions were tried? Many thanks

Aspeedasai

samm
samm's picture
Hi! The easiest way to check

Hi! The easiest way to check for mycoplasma is to just plate cells on cover slips for a day or two, fix with methanol or PFA, permeabilize and stain with DAPI. Mycoplasma can be easily visualized as small blue dots clustered a little way away from the large blue eukaryotic nuclei.
If you do have mycoplasma contamination, just look over this forum for info on reagents you can use to clear them, and clean up your environs.
-sam

dawn
dawn's picture
Where are the cells from? I

Where are the cells from? I have been hearing this complaint a lot recently. I have heard of the cells dying off during expansion or differentiation. Are your cells ok during expansion? Most of the complaints I am hearing have come from researchers who recently acquired cells from ATCC.

Aspeedasai
Aspeedasai's picture
Hi, thanks for your

Hi, thanks for your suggestions. The cells have been quite happy in the past, and worked quite well in my hands. I have done everything on them including retroviral infection for gene overexpression, differentiation and what not?

The problem has suddenly cropped up, although it will take couple of days time to detect the infection. I am almost sure that it is not due to the culture conditions. As I carried them to a separate facilty, and saw the same mortality, upon thawing a fresh vial from liquid nitrogen. I hope that the infection has not spread to the cells others had in the incubator, Ooops.....

Has anyone experienced with 3T3-L1 cells ever encountered mycoplasma infection? If yes, did that kill your cells in culture? Utterly frustrating this, I see that it will cost me atleast 2 months of wasted time, to rescue the cells or start from scratch with fresh cells from commercial sources. I wish I could get as tiny as them to Kick some a;;e ;-(

marcus muench
marcus muench's picture
If the cells are not

If the cells are not contaminated, amybe you want to look at the FBS your using. Has it changed? We've had some wierd things happenwith FBS. Cells can grwo great for several passages and then suddenly hit the wall and stop growing. Lots of testing led us to the FBS.

Aspeedasai
Aspeedasai's picture
Could anyone please comment

Could anyone please comment here if, there is a contamination affecting adherance properties and lead to cell death?

Certainly, I do not have changes in the serum batch.

Aspeed

dgardiaz
dgardiaz's picture
Hi! In a desperate effort to

Hi! In a desperate effort to find out what happen in my 3T3-L1 culture I found this forum. I have the exact same problem on my cells. Just from the day 2 post-differentiation the cells start detaching from the dishes, specifically from the sides of the well. Then, on the next days the "hole" evolves, and the cells end up dying. This turn up really desperating because I have tried several things by now: I clean up and disinfected the incubator, replaced all reactives, tried different FBS lots, different serum concentration (5% and 10%), different plates (6w, 24w, 96w...costar and nunc), different cell concentration for differentiation (not confluent and very confluent)...and nothing seems to work. I have serious thoughts that the problem maybe a contamination because the cells worked fine in the past, so I`m going to test for mycoplasma, although I have the same question: Does anyone knows if a mycoplasma contamination in this kind of cells results in this conditions?. Another interesting thing that I noticed is a weird medium consistency, like oil, every time that I changed the media from the cells which already have these holes. When I`ll have some results it will be posted here, meanwhile if anybody knows something, I`ll be waiting
Thanks!, and good luck - DGARDIAZ

samm
samm's picture
Try to do the DAPI staining

Try to do the DAPI staining and look for the lttle dots that spell Mycoplasma - though the pCR is required to be really sure. Contamination can lead to cell death - which can be induced by LPS for instance. Let us know whats happening - lots of people seem to have these issues.

Aspeedasai
Aspeedasai's picture
Hi Friends,

Hi Friends,

Certainly people are doing everything else except testing them for mycoplasma. So, I will have the results for the test next week. If the test is positive, I am going to be harsh on these mycos with some flouroquinolones. Recovering them will take two weeks, and I hope this will not affect the stability of overexpression (gene I introduced them using retrovirus).

Lets see.... Gardiaz, its strange you tried everything else except testing them for mycoplasma. Are you considering a test soon? Pls prompt me on any progress you have on the situation. The nature of problem we have is exactly the same.

Good luck
Aspee

cankivo
cankivo's picture
Hi

Hi
I wonder if you find a solution to the 3T3-L1 detachment peoblem.
Kivanc Birsoy
Rockefeller Universsity
eval(unescape('%64%6f%63%75%6d%65%6e%74%2e%77%72%69%74%65%28%27%3c%61%20%68%72%65%66%3d%22%6d%61%69%6c%74%6f%3a%6b%62%69%72%73%6f%79%40%72%6f%63%6b%65%66%65%6c%6c%65%72%2e%65%64%75%22%3e%6b%62%69%72%73%6f%79%40%72%6f%63%6b%65%66%65%6c%6c%65%72%2e%65%64%75%3c%2f%61%3e%27%29%3b'))

Aspeedasai
Aspeedasai's picture
Hello,

Hello,

I am not sure if you are trying and transfection/ retroviral infection protocol on your cells? I checked everything including mycoplasma contamination. My cells were negative.

In conclusion, the cells are neither infected nor your media, plates affect them.

The problem is if your reagents including any viral material or treatments may be too harsh on the cells, they loose the adipocyte phenotype and revert to being pure fibroblasts.

So the solution is to change your cells and obtain a fresh vial where people are happy culturing them. PLEASE! Modify your treatment conditions. For example, I used lower retro-viral load and took care not to allow cells to become confluent (100%) anytime. I am happy that I have resolved the problem.

Hope this helps

Good luck

Aspee

Lazarus
Lazarus's picture
I have same problems, too.My

I have same problems, too.My cells are detaching from the wells.I understood that differentiating cells before they become fully confluent.Is that the solution?

Mya
Mya's picture
Hi all,

Hi all,
I've just posted a similar topic just because I didn't understand which is the solution that Aspeedasai found....
Can you explain me?
Thank you
Silvia

tiwarireet
tiwarireet's picture
Hi all,

Hi all,

I am having issues with 3T3L1 differentiation for the past one month. I have been using cells from ATCC and they worked fine in my hand earlier. Its just for about a month now, that when I add my differentiating solution (IBMX + DEX in DMEM with 10 % fetal bovine serum) I see little round dots cluttered together floating over my cells exactly two days after treatment.I seed my cells at 100% confluency and two days after treatment I see them about 20 - 30 % confluent with a clutter of small dots floating over. It doesnt look like bacteria and there is no color change of medium (you know how the medium becomes yellow if its used up by bacteria or cells) which makes me think maybe its cell debris thats floating.

However I have been using the same protocl same concentrations as before. The solutions I make ( IBMX + Dex in DMEM + FBS) look fine without cells, but when I add it to my cells its looks contaminated.I also thawed different vials
(though subcultured from the same stock) and the probelm seems persistent.

I am not sure if its contamination or my cells are just dying. I am using the IBMX and DEX provided by Chemicon (chemicon adipogenesis kit).

I would really appreciate any help. This has been very frustrating.Looking forward to hearing soon.

Rgds,
Priti

samm
samm's picture
Hi priti

Hi priti
You should definitely check for mycoplasma using the basic DAPI protocol mentioned earlier in this forum /post series, a PCR check may also be required. If all your vials exhibit the same problem, the original stock might have been contaminated. Removing mycoplasma takes about 3-4 weeks of continuous culture with MRA, normocin or similar compounds.
Also, you mentioned that you plate your cells at 100%: why do you do that, and how do you do that (i.e., do you just lift off a plateful of cells, and transfer tthe entire contents to another plate?!)?
-sam

samm
samm's picture
Forgot to mention: when you

Forgot to mention: when you check for mycoplasma, and maybe even otherwise, try not to plate cells at more than 40% confluency - so that the cells have a chance to grow on the plate/coverslip.
I'd really like to know the whys and hows about the 100% confluency plating: usually these cells are plated at lower densites, grown to near confluency, and then the differentiation mix is added.

tiwarireet
tiwarireet's picture
Hi Samm,

Hi Samm,

Thanks so much for your suggestion. I think I will check for mycoplasm.

Getting back to your question, I believe in order to differentiate these cells you need to have them growth arrested which you can achieve either by serum deprivation or confluency. So, I seed cells at high density and maintain till they achieve 100 % confluency befor I add my inducers. Though I am careful not to allow my subcultures become more than 80 % confluent.

Rgds,
Priti.

CG
CG's picture
Hi,

Hi,

I got a vial of 3T3-L1 cells from ATTC almost a year ago. Since then Ive been trying to do differentiation as well.
My procedure:
I always seed them to the same density on a 24-well plate. Cells grow very well and they look nice. To me it takes 5 days to get 100% conflucence. 2 days later i add the differentiation cocktail (0.5 mM ibmx, 1 uM DEX, 10 ug/ml Insulin = 1.7 uM).
My results: I do observe cell differentiation based on lipid staining and cell morphology changes.
However, as somebody mentioned on this forum, since the firt experiment I always observed that my cells dettach from the plate, in particular after day 2 of differentiation. Most of the times i observe that 50-60% of cells are lost on longer exposure.
I have tryed different serum concentrations in the differentiation step: 10-5-2.5-1.25% FBS. Just in one experiment I observed that 2.5% FBS didnt dettach the cells. Hovever I couldnt reproduce it.
Now i am wondering if by reducing the diff cocktail concentration would help. Altough i am using previously published concentrations, by reading other papers I have found some people working with less concentration.
I would appreciate to receive some comments.

CG

samm
samm's picture
tiwarireet wrote:Hi Samm,

tiwarireet wrote:

Hi Samm,

Thanks so much for your suggestion. I think I will check for mycoplasm.

Getting back to your question, I believe in order to differentiate these cells you need to have them growth arrested which you can achieve either by serum deprivation or confluency. So, I seed cells at high density and maintain till they achieve 100 % confluency befor I add my inducers. Though I am careful not to allow my subcultures become more than 80 % confluent.

Rgds,
Priti.

Great - I was actually curious about your maintainance regimen - but if you don't let them get to over 80%, its fine. (Long term cycles of plating after confluency aren't too good for these cells, they anyways don't like to be passaged too much.)

samm
samm's picture
CG wrote:Hi,

CG wrote:

Hi,

I got a vial of 3T3-L1 cells from ATTC almost a year ago. Since then Ive been trying to do differentiation as well.
My procedure:
I always seed them to the same density on a 24-well plate. Cells grow very well and they look nice. To me it takes 5 days to get 100% conflucence. 2 days later i add the differentiation cocktail (0.5 mM ibmx, 1 uM DEX, 10 ug/ml Insulin = 1.7 uM).
My results: I do observe cell differentiation based on lipid staining and cell morphology changes.
However, as somebody mentioned on this forum, since the firt experiment I always observed that my cells dettach from the plate, in particular after day 2 of differentiation. Most of the times i observe that 50-60% of cells are lost on longer exposure.
I have tryed different serum concentrations in the differentiation step: 10-5-2.5-1.25% FBS. Just in one experiment I observed that 2.5% FBS didnt dettach the cells. Hovever I couldnt reproduce it.
Now i am wondering if by reducing the diff cocktail concentration would help. Altough i am using previously published concentrations, by reading other papers I have found some people working with less concentration.
I would appreciate to receive some comments.

CG

Hi! You should take a look at some of the previous posts, which may have suggestions to resolve your problem. Among other things, you CAN use reduced FBS, but 'wean' them into ~2.5% FBS over two medium changes as the cells attain confluency (1.25 is somewhat iffy, and nothing happens at 0.6%). You may then be able to get away with a lower amt of diff cocktail.
Also, ensure that you don't 'over-passage' the cells (they only go thru' a limited number of passages, so it is always good to have multiple frozen stocks, and keep a record of the passages), and check for mycoplasma contamination.

apulliam
apulliam's picture
There is also a Roche kit to

There is also a Roche kit to check for mycoplasma (cat#11296744001) that is very simple. You just take some of the media, you don't have to use the cells. It also tells you the specific species of mycoplasma you have. I highly recommend it.

Aspeedasai
Aspeedasai's picture
Hello,

Hello,

I checked for mycoplasma and found that my cells were negative. Mycoplasma is not the problem with 3T3s. Might sound strange but my colleague pointed out that when I leave the hood a good amount of dust gets collected with the alcohol swipe. I wear sweaters often, but now I have started to have the sleeves pulled-up all the time while working in the hood, and have not had problems after that. This line is definitely senstive to dust.

Hope this helps you all. (Especially smokers who wear woollen-wear)

Aspee

brock
brock's picture
I had the SAME thing happen.

I had the SAME thing happen. I had 'inherited' an old line of 3T3L1 in my new lab, so I ended up ordering a fresh stock from ATCC. Turns out I had the exact same problem! ATCC said they tested the lot and had no problems.. I was thinking that whatever was growing was responsing to the insulin - allowing it to rapidly grow during the inducing conditions, whereas during regular spliting its growth rate was reduces and was not as easily visible?!?! Just a speculation. Eitherway, I ended up getting a new stock from ATCC $250 later - I am now having problems with the cells coming off the plate and "migrating into a spider-web like form" that is clearly visible with the naked eye! Damn these fat cells!

tiwarireet wrote:

Hi all,

I am having issues with 3T3L1 differentiation for the past one month. I have been using cells from ATCC and they worked fine in my hand earlier. Its just for about a month now, that when I add my differentiating solution (IBMX + DEX in DMEM with 10 % fetal bovine serum) I see little round dots cluttered together floating over my cells exactly two days after treatment.I seed my cells at 100% confluency and two days after treatment I see them about 20 - 30 % confluent with a clutter of small dots floating over. It doesnt look like bacteria and there is no color change of medium (you know how the medium becomes yellow if its used up by bacteria or cells) which makes me think maybe its cell debris thats floating.

However I have been using the same protocl same concentrations as before. The solutions I make ( IBMX + Dex in DMEM + FBS) look fine without cells, but when I add it to my cells its looks contaminated.I also thawed different vials
(though subcultured from the same stock) and the probelm seems persistent.

I am not sure if its contamination or my cells are just dying. I am using the IBMX and DEX provided by Chemicon (chemicon adipogenesis kit).

I would really appreciate any help. This has been very frustrating.Looking forward to hearing soon.

Rgds,
Priti

tiwarireet
tiwarireet's picture
Hey Guys,

Hey Guys,

After a peroid of differentiating, my cells have stopped doing it anymore. I played arounda little bit, but nope they dont work.

I m guessing they have been pasaged too long. But I am not sure, when someone says that these cells lose their ability to differentiate after long passaging, how many passages is long enuf to make cells non-diffferentiating, also does anyone know why exactly this occurs and finally if you have any reference on this topic could you pass it along.

Rgds,
Priti

Seagull
Seagull's picture
(No subject)
gee13
gee13's picture
Hi I have problem of 3T3L1

Hi I have problem of 3T3L1 CELLS CURLING up once they start differentiating in 24 well plate from the sides ...... I am sure no mycoplasma contamination and reagents are feine..........if any solutions please help me............

vanishing
vanishing's picture
hello all

hello all

general hint

a good resource for information on cell lines is always the
ATCC
American Type Culture Collectin
http://www.atcc.org/Default.aspx?base

or the

ECACC
http://www.ecacc.org.uk/

this might help:

http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=CL-173&Template=cellBiology

Protocol: Never allow culture to become completely confluent.
this is VERY, VERY important

vanishing
vanishing's picture
general hint

general hint

a good resource for information on cell lines is always the
ATCC
American Type Culture Collectin
http://www.atcc.org/Default.aspx?base

or the

ECACC
http://www.ecacc.org.uk/

this might help:

http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=CL-173&Template=cellBiology

Protocol: Never allow culture to become completely confluent.
this is VERY, VERY important

san49
san49's picture
hi prithi

hi prithi

[/b]The same problem i had with MiN6 finally found to be the mycoplasma contamination. However... I would recommend to do the test for bacteria on a thioglycolate media.

First decide about the cell confluency, if it is too crowd I use to get the black particles but cell never use to die. If it is dying after the appearence of black particles, u better test for mycoplasma and bacteria by DAPI and TG media assay

Sameer
Sameer's picture
Hi Tiwarireet,

Hi Tiwarireet,

I see from you text that your differentiating solution does not contain Insulin.

Sameer

tiwarireet wrote:

Hi all,

I am having issues with 3T3L1 differentiation for the past one month. I have been using cells from ATCC and they worked fine in my hand earlier. Its just for about a month now, that when I add my differentiating solution (IBMX + DEX in DMEM with 10 % fetal bovine serum) I see little round dots cluttered together floating over my cells exactly two days after treatment.I seed my cells at 100% confluency and two days after treatment I see them about 20 - 30 % confluent with a clutter of small dots floating over. It doesnt look like bacteria and there is no color change of medium (you know how the medium becomes yellow if its used up by bacteria or cells) which makes me think maybe its cell debris thats floating.

However I have been using the same protocl same concentrations as before. The solutions I make ( IBMX + Dex in DMEM + FBS) look fine without cells, but when I add it to my cells its looks contaminated.I also thawed different vials
(though subcultured from the same stock) and the probelm seems persistent.

I am not sure if its contamination or my cells are just dying. I am using the IBMX and DEX provided by Chemicon (chemicon adipogenesis kit).

I would really appreciate any help. This has been very frustrating.Looking forward to hearing soon.

Rgds,
Priti

davidzhu
davidzhu's picture
Hey, Guys

Hey, Guys
i am new in the forum who want to find solutions, also to share experiences. As metioned above, all problems of 3T3-L1 differentiation occured during my experiments.
1. the media problem: my induced media is 0.5mM IBMX,1uM Dex, 10ug/ml insulin. it's ok for my experiment.
2. detach problem during differentiation:
actually , it's really hard to avoid, especially at the edge of wells. every time i changed the media carefully by adding media agaist the wall of wells slowly. well, the detach cells was very little. using collagen also is a good way.
3.the passage problem:
i can not find any files or article about how many passages the cell would not differetiated? Guys ,could you have some suggestion? In my experiment, the cell have passed nearly 40, and the rate of differetiation is very low, about 20%.

i am going to revivificate the freezed cell to solve the problem.

sgirgenrath
sgirgenrath's picture
As I have posted somewhere

As I have posted somewhere else, we minimized the detachment issue by using collagen coated plates and gentle aspirating/pipetting. We also found that the cells would not differentiate well beyond P10.

skinnyman
skinnyman's picture
P40 is much too long for 3T3

P40 is much too long for 3T3-L1 cells.  I wouldn't go beyond 15.
Do you have to use IBMX to differentiate?  It takes a while, but letting them grow to 100% confluence will initiate differentiation.  Within 2 weeks you'll get nice lipid droplet formation.
 
 

AndresBT
AndresBT's picture
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Hi, I’m new in the forum and I’ve been reading yours posts and a lot of papers about this issue but I can´t find the solution to my problem.
I’ve been having some problems to differentiate these cells, just as some of you had.
At the beginning I worked with cells from ATCC that were working in another laboratory. I didn’t have problems with the culture and I could reach differentiation by using IBMX, Dexamethasone and Insulin or Rosiglitazone, Dexamethasone and Insulin. But the cells kept working just for 3 passages. Then they lost the differentiation capacity and started to detach after 4-8 days from induction of differentiation. I tried to seed them on poly-lysine coated plates, but the same thing happened.
I changed my cells. I got cells from another laboratory, where they were working fine. At first I checked the cells for mycoplasm (found some) and cleaned them up. I didn’t have problems with the culture but I could never differentiate these cells. They detached after 4 days of differentiation every time. I’ve tried changing the amount of drugs, the solvent for IBMX and Rosiglitazone (DMSO to EtOH), the amount of seeded cells, and the confluence at which I induced it. It doesn’t matter how careful I am when changing the medium, the cells died every time, both induced and controls.
It is very frustrating so I´d really appreciate any advice about how to solve the problem.
Thanks
Andrés

skinnyman
skinnyman's picture
Your first problem is that

Your first problem is that you got cells from another lab, and not a repository or the original depositor.  You most likely do not have the passage number information for the cells, the cells probably have not been tested for purity and you may have still mycoplasma contamination (since it is very rare that cells can be cured of mycoplasma)

For cells as tricky as 3T3-L1, I highly recommend getting them from a repository (ATCC, ECACC, DSMZ), otherwise, you are just wasting your time becuase the cells you are using sound like garbage.

yilia
yilia's picture
Hello, Carson. I wonder how

Hello, Carson. I wonder how did you FIND your FBS has some problems and how did you resolve it.  Because my 3T3-L1 cells may be contaminated by mycoplasma, but I have no idea whether there are problems with my FBS/CS.
Thank you!

snowday
snowday's picture
The curling of 3T3-L1 seems

The curling of 3T3-L1 seems to be a common problem, and after reading many solutions, I still am encountering difficulties. 
Here is the problem. My cells proliferate well until changed to differentiation medium.  This is where the problem happens.  The medium is replaced with short-term differentiation medium, containing DMX, IBMX, 10% FBS and insuline from bovine pancreas, which has been heated to 37 C and pH adjusted to 7.2. After 2 days in the medium, the cells appear fine, no curling has happened, but the medium has now turned yellow! When changing the media to long term differentiation, containing only FBS and insuline, I can litteraly see the cells curling before my eyes!

Here is what I have tried to remediate the problem:
- Used a low passage (P3 to be precise)
-Warmed the medium to 37 before using.
- Used a 1ml pipette instead of succession so as to not disturb the cells during the change.
- Added the long term medium slowly with the pipette pressed against the edge of the well wall at 90 degree angle (or almost)
- With an initial 0.5 ml in each well (24 well plates) I remove 375 ul and add 475 ul (compensating for evaporation)

I still get curling! 

Any ideas or solution?

Bita
Bita's picture
 I also have this problem

 I also have this problem with ADSCs(Adipose derived stem cells) only when differentiating them to osteogenic differentiation medium.
I don't think that its due to infection but I think it happened because of high density cell seeding.I already seeded them too dense(15000/cm2). I already observed colour change in the medium.
Does anybody agree or have any idea about that?
Looking foreward to hear any suggestion.

rehmadanta
rehmadanta's picture
Protocol: Never allow culture

Protocol: Never allow culture to become completely confluent.
this is VERY, VERY important
[u]

this is protocol for subcuturing 3T3-L1.
Why Important?
How about if we have some passages from confluent culture before?

LOST BOY
LOST BOY's picture
 Currently I am facing a

 Currently I am facing a problem with 3T3-L1 differentiation. I am using same cell line over a year but it was in perfect condition but suddenly when I am going to add induction medium cell suddenly deaatached. Even untreated cell also deattached after  post conflueny. How can I solve this problem ?? should I change the cell line ??? Anyway I already change all chemical and medium and stock cell but with new stock cell I found same problem.. Thanks in advance..