Hellow. I'm doing deal with MC3T3-E1 osteoblast (ATCC) for the experiment.
It has been quite a long time to culture this cell.
However, something started getting strange.
It was just very healthy and easy cell to culture. But after that time, the cell couldn't grow well.
1. I usually got a supply of this cell from other labs. When I thaw this cell, the morphology is ok just after thawing, however, it started getting strange or dead after 2 or 3 times of subcultures.
(This is strange because I use the same protocol for every cell types, but other types are very fine.)
2. Also, after 1 or 2 subculture (before cell morphology is strange) I stocked this cell, and after some time when I thaw this cell, it is just dead. (It can't adhere to the dish)
I changed many things such as an incubator, Trypsin working time, Media product, FBS..
This phenomenon is going on.
Other types of cell are very fine without this cell.
My research is stuck for 6 months..
Please somebody advise me.
Thank you.
PS. Do you think it is a good idea to purchase a new MC3T3-E1? Because I have been got this cell from several other labs. But these labs said they are fine in their labs.
Hello Hyerim.
It sounds that your basic cell freezing and thawing may have problem in the procedure. Check your procedure for the rates of cell survival and growth before cell freezing and thawing in your osteoblast stem cells and other somatic cells, i.e., CHO or other cells running together whether you are sure that you freeze and thaw cells properly. Study cell freezing and thaw in a standard cell culture textbook protocol that gives all the pitfall and considerations. The protocol may be simple to follow. However, without understandable cryobiology knowledge and experiences fully it happens in every biology core lab, not too mention of engineering labs.
Follow the protocol after making all solutions fresh and sterile with fast working and concentration. Determine cell death and subsequent culture of the frozen-thawed cells with propidium iodide or equivalents or trypan blue after the F&T procedure. If you cannot find any problem with your procedure in your hands, change the cell stock from commercial sources. Your cells are stem cells and tend to differentiate when you overgrow, causing acidic condition, or no close supervision is around over the cultured cells to tell you 'It's OK'.
Regards.
Hi Hyerim,
One of the first things that come to my mind is mycoplasma contamination. Since you get your cells from other labs, it is really important to check whether they are mycoplasma negative. There are commercially available kits to check mycoplasma contamination such as the one from Lonza: https://www.lonza.com/products-services/bio-research/cell-culture-products/mycoplasma-detection-and-removal/mycoalert-mycoplasma-detection-kit.aspx
If the problem is not freezing/thawing as Dr. Sang Ho Lee mentioned and if you are sure about your protocols, your reagents such as DMSO, FBS, medium, etc., you can check for mycoplasma contamination. Another reason can be the passage number. Cells in very high passages will enter senescence and this will cause the problems you encounter.
And if the cells are at high passages or mycoplasma-positive, definitely, you should buy a new vial. You also mentioned that other labs say their cells are OK. Do you use exactly same reagents or flasks?
I hope this info helps and you can find what the problem is. Good luck with your experiments!
Thank you, Mr. Lee and Sezin. I'm going to consider your advices and figure out what is the problem. Than you!! :)
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