Hi fellow researchers,
I have been using this MSC cell line (hTERT immortalised) for some time. However, they have very different growth pattern (See attached) and I have done test on what factors might affect that (passage number, cell density prior to seeding the cells etc.) but came back empty handed. The pattern seems to appear completely random. Usually the same pattern can be seen if they are plated at the same time. The ideal pattern would be cells growing fully confluent, instead of looking like a web.
I am just wondering if anyone out there might have seen something like that before?
Procedure:
2500 cells were seeded in each wells on a 384-well plate. They were grown for 5 days in AIM-V serum free medium (it's a co-culture system and if I add the target cells and the drugs the MSC would have been growing for 5 days).
Thanks a lot!
How do you passage these cells? Sometimes tripsin makes some damage and cells become stressed. I usually use low concentration tripsin, 0.05% for five minutes. At the beginning I used 0.25% for 1 minute but then I realized that sometimes cells got stressed and they didn’t grow well. There were changes in the morphology as well!
Diego Raffo That is the next thing I want to test. I follow a protocol from the lab that sent us the cell line: 10 minutes in trypsin, but they did not specify the concentration. We have been using 0.25%. Perhaps I should shorten the trypsinising time or lower the concentration. This is very possible especially with the serum free culture I do in the co-culturing. Any carryover won't be neutralised by the medium.
For what you mention tripsin is probably the problem! What do you use intead of serum? Platelet lysate also inactivates tripsin! If you are using a mediun without serum or platelets you may consider changing tripsin by tryple of thermo fisher that is lighter. Also you can buy tripsin inhibitors, a believe Sigma have an option for that.
Diego Raffo We just use AIM-V medium designed for haematopoietic cells because we are doing drug screening on leukaemic cells. It never was a problem with our collaborator so we never really think about what trypsin could do in the system. Probably for now I will just wash it again with complete RPMI before seeding them. Hope it would improve the situation enough to save us the hustle and money. Thanks Deigo!
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