Dear Dmitri,

I address you to our papers:

Kadyshevich, E.A., Ostrovskii, V.E.: Hypothetical physicochemical mechanisms of some intracellular processes: The hydrate hypothesis of mitosis and DNA replication, Thermoch. Acta, Vol 458, pp. 148–161, 2007;Ostrovskii, V.E., Kadyshevich, E.A.: Mitosis and DNA replication and life origination hydrate hypotheses, in: H. Seligmann (ed.), DNA Replication –Current Advances, InTech, Rijeka, Croatia, pp. 75-114,

2011 (both are available at the ResearchGate).

I am sorry, they scarcely will help you for searching an answer to your question but, may be, will force you to meditate upon the global mechanism of the cellular metabolism.

Best wishes,

Victor

Dear Dmitri

your study is quite interesting..by the way you didn't mention about the incubation period. As per my knowledge 3T3-L1 cells resemble adipocytes (rich in lipid droplets). when there is higher lipid accumulation in the cells with time, cell density related issues may popup (normally lipids float), which makes the cells to float. you have 3 ways to over come your problem, 1. reduce the incubation time so that cell does not confluent completely (70% confluence is enough for most of experiments) so that lipid content wont exceeds much. second methods is increasing the Ca2+, Mg2+ ions concentration in our media, which enhance the cell binding to the flask surface. third is coating your plates or flasks with fibronectin, etc. Hope your problem get solved..all the best

You either are using an old passage, or you seed them too confluent or your cells may have mycoplasma contamination.

Dear Dimitri

I advise you to recheck the condition of your cell culture, Medium, and specially pH and additives...and in parallel check the CO2 levels in your incubator.

So start with a new vial, new passage and new medium and in the same time do have a positive control in your incubator like a cell line that you know for sure that it works without having that peel off....

After this check list, and if the problem persists, you might need, due to the brand of Plate that you are using (might not be convinient for cell culture) to due some collagen or Polylysine coating prior seeding your cells.

Good luck

Hi!

I agree with Matilde's. I had the same problem before and found it was due to cell overgrowth. However, you may still use your culture even the layer is 'peel off'. Only thing you need to take care is not to break the layer and try to re-attach the 'peel off' layer back when you process the culture for staining..

I also agree with Matilde's response. I work extensively with 3T3-L1 cells and they almost always remain adherent. However, some passages of the cells do suffer from the 'peeling off' phenomenon that you have experienced. Usually this is specific to certain passages of cells, and it can be worsened by seeding the cells at too high a density. In our experience it is the IBMX in the induction cocktail that causes these passages of cells to detach.

If possible, try to obtain a new passage of cells from elsewhere. Hopefully this will solve the problem

Hi Dimitri,

I agree with the previous posts suggesting it could be a 3T3-L1 passage effect. I also remember reading on a number of other forums that certain batches of cells from particular companies were proving problamatic with this issue.

Thankfully I have never experienced the "peeling" effect of differentiating cells however I have had other egde effects before. Cells would sudenly die at the edges of 6 well plates and I put this down to periods during media changes where the edges would dry very quickly. Changing media of cells in batches and speeding up media changes has stopped all edge effects for me. Not sure if it will help your peeling situation but could be worth a try.

Buying new cells and looking after them is also a good idea to prevent wasting a lot of time "trouble shooting" these cells. We use 3T3-L1 cells from ZenBio and I would highly reccomend them, they differentiate very well and we have had no major problems with them.

Good luck with your problem, these cells can be a right pain sometimes!

George

Thank you all guys for your useful insights!

Hello Dmitri,

I was wondering if you resolves that peeling issue an how ?

Hi Petar, as some of the answers suggested here, I have tried to start new vials with a lower passage and tried not to keep the cells too confluent for a long time (not to wait more than 48 hours between the time the cells get 100% confluent and the time I start the differentiation cocktail). I also try to take extra care when I wash the cells and change medium while differentiating the cells. Another thing I have tried is to coat the plates when I replate the cells after the differentiation. I usually use collagen or gelatin.

I have implemented all these changes in my procedures and so far the cells are behaving well.

I hope it helps.

Let me know if you have any other questions.

Hi Dmitri, what do you mean by replating the cells after differentiation on collagen-treated surface ? I use very low passage number (ATCC +4), but I continue to obtain "perforation" and monolayer contraction/retraction (please have a look at the attachment), independently if I add the diff. cocktail upon reaching confluence or 24-28h later. It is my feeling that those cell have lost their contact inhibition, because when I do primary preadipocytes cell culture, using the same media and reagents, I don't get such effects. What do you think?

Petar, thanks for sharing your results with me. In my experience it is very important to be very gentle with these cells, they are so sensitive to any stress. When I wash the cells or change medium I make sure not to direct the flow from the pipet directly to the cells. Instead, I tilt the plate and push the medium out of the pipet really slow and on the side of the plate (P10 plate). I also do not use vacuum to aspirate the medium. I am sure if you follow these steps your cells will be much happier.

If you would share your differentiation protocol, I perhaps might suggest other improvements.

I think that the sensitivity of your cells to detachment is specific to the stock of cells that you're using, as in my experience this is not a general problem with 3T3-L1 cells. Although I have had some vials of cells that do detach badly during differentiation, usually this is not a problem. And when culturing the cells I am not gentle with them: I always use a vacuum to aspirate the medium, although when adding fresh media I direct the stream down the side of each well, rather than directly onto the cells (but I don't do this very slowly).

There could also be an issue with the type of cell culture plastic that you are using. We always use Corning plates and flasks. Although these are more expensive, in our experience they give more consistent adipogenesis, and better adhesion.

Be careful not to disturb the cell layer while adding medium, I know many ppl say they do take extra care while adding medium but more care may solve this prob if cells are not too confluent.

I agree with all of the passage, volume of medium, plastic quality and degree of confluency comments from above. 48h is the absolute maximum I would leave cells after confluency is seen. Even this may be too long. The reason this problem is most obvious at the edges is that charge is not distributed evenly over the flask/plate bottom making unhappy cells most likely to 'peel off' first at the edges.

Passage number and plastic quality I could see having an effect but I would have to disagree with the length of confluency comment above. I have held 3T3-L1 cells at 100% confluency for upto 16 days without observing any of the edge effects you have described. To be fair I did not try to differentiate these cells as they were used as a -ve control but they were happy enough. This cell line requires at least 48 hours at 100% confluency in order to allow for growth arrest to occur before differentiation should be initiated. For me it is an issue with the stock cells you are using.

Fair comment with regards to requirement for 48h at 100% confluency before growth arrest is seen, but do you not start to see 'balling up' of cells within the culture during this 48h and free cells starting to appear? I have always taken this to be continued cell division with nowhere for the cells to go! So exactly what you say: no growth arrest despite confluence. If cells are less than happy they might start to detach at this point and the edge of the culture is probably where it shows most. I guess we are all saying multifactorial?

I have never observed what you describe, this cell line should be contact inhibited and therefore prevent any further cell division from occurring. This may be a result of too many passages, how high a passage number do you use for this cell line? An alternative might be that during propagation, when trying to increase the number of cells, cultures may have become too confluent and then continued to be split and used for later experiments. This however usually just results in poor differentiation. Like you say there are many possibilities as to what might be reason. If you are having problems though, I would recommend obtaining a new batch of cells from a reputable company.

Mustn't forget mycoplasma!

I have spent a lot of the last year working with 3T3-L1 cells and often have this problem. I have found the higher the passage number the more likely this will occur. It is not a symptom of mycoplasma in this case, they just tend to become loose the older they get. Keeping passage number low definitely helps where possible, ensuring your preadipocyte stock flasks never grow more than 70% confluent seems to help as well. Being gentle with your pippetting also helps enormously; cutting the ends off plastic pippette tips to widen them and generally pippetting very slowly for me has basically eradicated this problem.

*You also must remember that during adipogenesis, these cells undergo mitotic clonal expansion (MCE) and re-enter the cell cycle to undergo another 2 rounds of mitosis before expressing the full adipocyte phenotype - so your samples will be over confluent by the end

I agree- the brand of culture plastic can have a large influence o n cell attachment. I also use only Corning for the same reason as William for. We have had several cases of particular cell lines doing much better on Corning than on other plastics. Once you have a plastic your cells like don't change it for something cheaper! Or borrow something different from another lab.

Also are you feeding the cells regularly as per protocol. They will lift off under nutrient stress

Need to use lot less suction. Peeling off is very common in these cells. Just pipette the media off with regular 10ml pipettes.

I haven't had success with 6-well and 12-well plates for 3T3 cells. They start to detach. I am using corning flat bottom non-pyrogenic,polystyrene ones. Is there any recommendations?

Someone has used Poly L Lysin? for attachment the cells

Hi Francisca Villavicencio Tejo . Did you try the poli-L-lysine coating? I will try that tomorrow... and want to know if you have any issues. Also Dmitri Samovski , do you remember what was the collagen concentration you used? I have healthy 3T3 L1 cells which perform great in the undifferentiated state and also differentiate well. My problem is when I will do the Oil Red staining and cells easily dettach with the washes...

Hi everyone, I've just started to work with 3T3-L1 cells and I have arrived at this thread because I am having the same problem. When in 96-well Greiner cell culture treated plates at confluence, these cells lift off after 2 HBSS washes. I am treating with 100 uM carboxy-H2DCFDA in order to perform a ROS assay, and treated cells completely detach after 2 washes (or 3 if you count administration of the probe). However, my untreated cells detach only around the edges so the carboxy-H2DCFDA is definitely having a "detaching effect". I would be curious how the poly L lysine coating works (?).