Hello everyone. About three months ago, I could detach my mesenchymal stem cells obtained from 8-week-old mice with trypsin %0.25 on the 10th day in 3-4 minutes. However, now I can not detach the mesenchymal stem cells with trypsin %0.25 that I have obtained from 8-week-old mice in 10 minutes, even on the seventh day. At first, I used the "Gibco™ Fetal Bovine Serum, qualified, heat-inactivated, Brazil" serum in the medium of the cells I used. Now I use "Gibco™ Fetal Bovine Serum, qualified, heat-inactivated, United States" serum in the medium of the cells. Can different serums cause such different results? Or is there something I do not know? I would really appreciate any help.
I believe serum will not cause this. May be, change Trypsin lot. Try to borrow Trypsin from Neighbour lab or buy from a different company. Good luck
Hello Muhammed Burak Batır
While in culture, there is a probability that the mesenchymal stem cells could often have their gene expression altered and the expressed proteins may get modified, resulting in cells undergoing unwanted differentiation.
A number of studies have been carried out in order to understand and control changes in cell culture, and certain assumptions have been raised such as,
1) The presence of fetal bovine serum in cell culture medium is questionable since there is a lack of characterization of these substances (especially the growth factors), and the amount of such substances as well. The ideal culture media should contain standard nutrients in accordance with the cell type and necessity.
2) When you use proteolytic enzymes during subculturing it may promote the destruction of both the extracellular matrix and surface proteins and may, thus, modify signaling and mechanotransduction of signals to the nucleus.
So, spontaneous differentiation of mesenchymal stem cells can occur during expansion in culture likely to be caused by inappropriate culture conditions. This can lead to very adherent differentiated cells.
I suggest that for cell detachment you may try mechanical detachment using cell scrapers to harvest cells cultured in flasks. Although a cell scraper-based method is simple, the percentage of alive detached cells may be affected. You may also try using another method namely, combination of EDTA and chilling. You wash the cells with cold PBS + 2mM EDTA two times and then place the cells in cold 2mM EDTA for 20 mins.
You may also want to refer to the paper attached below. It may be helpful.
Article The likely role of proteolytic enzymes in unwanted different...
Best.
May be the problem is with the trypsin. If it is freezed thawed many times, its strength is reduced. You have to aliquot the trypsin in small volumes, just necessary for 1 day. When you need, you take 1, the other aliquots should remain safely in the freezer. Each time you need, take 1. So the trypsin is only subjected to 1 freeze thaw cycle. The more it is freeze thawed, or the longer time it is kept in 4 degrees in the fridge, its strength is reduced and you need longer time to detach your cells.
Also you have to pay attention, before giving trypsin, you should wash the cells thoroughly, as no serum remains. Serum has antitrypsin activity.
In my lab, we now use TripLE select, instead of trypsin, it can be stored at room temperature, so no need to aliquot. Trypsin should be kept frozen, so you need to aliquot.
Many thanks for the answers. We washed our cell 3x with PBS and used acutase for detachment of the cells. Problem is solved.
It may be becuase of the culture medium. I've also had issues with mesenchymal stem cell cultures in medium supplemented with FBS. Making a good wash with PBS removed any traces of FBS and solved the problem. Also, using TrypLE by Gibco helps since its reccomended for some mesenchymal stem cell lines.
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