It is not a great idea to let these cells get too confluent, they have a tendancy to die off if they are left too long without being fed, and fail to thrive after splitting, as you have observed. In my experience these cells should not be left to confluence except in an experiment, at which poin a confluent culture needs to be fed every other day. Also amphoteracin can be cytotoxic to osteoblasts, maybe cut this down or out.

Good luck with your future experiments!

At this point, I can suggest only one thing - use 0.1% gelatin or collagen coating to help the cells attach. Have you checked for mycoplasma contamination? Alternatively, try Nunc brand plates.

Greetings:

1. Some alpha mem medias include ascorbic, which push the cells to differentiate. Double check this.

2. Try splitting at 80% confluence, instead of waiting for 100% confluence.

3. Try seeding a slightly higher density (1:5, 1:4).

4. How often are you changing the media and how much media are you using in the T75? 10-15 mL of media in a T75 changed every other day should be enough.

Have you checked the "too obvious to be possible" things, like is your CO2 low/out, or is the incubator/humidity off/ technical issues?

It is not a great idea to let these cells get too confluent, they have a tendancy to die off if they are left too long without being fed, and fail to thrive after splitting, as you have observed. In my experience these cells should not be left to confluence except in an experiment, at which poin a confluent culture needs to be fed every other day. Also amphoteracin can be cytotoxic to osteoblasts, maybe cut this down or out.

Good luck with your future experiments!

Thanks all! Your advice was very helpful and was corroborated by other experts.

Recently, I start having the same problem, the cells (MC3T3-E1) do not adhere to tissue culture flask. I admit that when I proliferate the cells, I waited until they formed 100% confluent layer. However, I was able to do several experiments after I proliferate them, then they stopped adhere to the tissue culture flask. After 8 hours, I can see them under the microscope survive, shine cells, floating in the media, yet they do not adhere. I am wondering if that is the main issue.

I have a quick question please, for how long can MC3T3-E1 be stored at -80 refrigerator?

Also sometime when these cells come out of the liquid nitrogen they take a while to recover, so what I do is put about 10ml of FCS into a T75 the day before I am going to thaw the cells out and leave the flask at 37C overnight.  The following day I aspirate the serum and thaw the cells out and plat ether and get much better recovery.  You could always try adding collagen, but this will probably push OB differentiation a little bit, but worth a go.

Good luck!!

I faced the same problem, you'll have better luck if you've some frozen cells. Thaw them, start anew. Those floating cells are useless.

I have used corning and Nunc brand plates and flasks to culture MC3T3 cells in ATCC recommended media (Alpha MEM, 10% FBS and 1X Pen/Sterp) and culture conditions and it is working fine for me...

You should subculture the cells when they are 70 to 80% confluent, do not wait for 100% confluency until required for some experiment.

Can I use DMEM-high glucose media instead of alpha MEM?

Hi Juhi

It is always better to use ATCC recomended media for particular cell lines. For MC3T3 ATCC suggest Thermofisher Alpha Mem with no ascorbic acid. Check ATCC website

cultural method for MC3T3 page for further clarification.