Trying to isolate MSC from mice/ rat bone marrow. Upto second passage cells are proliferating and in good morphology. When I am trying to expand the cells (second passage) cells are not attaching or they are dead. None of the culture conditions were changed fro first or second passage except the flask area (T25 to T75). Please give some advice for this problem.
Add 50 microM of beta-mercaptoethanol (final concentration) into the media. This will solve the problem.
Add 50 microM of beta-mercaptoethanol (final concentration) into the media. This will solve the problem.
Possibly something wrong with technique of passage. Check pH, dilution factor and concentration of all used solutions, type of used T-75.
Cell should be without media as minimum as possible. The pH could changes very fast in common medias during exposure to air. So be sure of addition HEPES in it.
Alternative possible reasons: CO2 level, mycoplasma contamination, long storage of FBS after defrost, too long period of propagation with very small starting concentration of cells, order of steps.
If your cell were round after tripsynization they was alive before, if not round then - dead. It could be helpfull for identification of time then cells died.
Good luck!
When I used beta mercaptoethanol my cells were surviving but showed morphology similar to astrocytes...
Hello
Yes , add 50 microM of beta-mercaptoethanol or try expand MSCs by plating at 50 cells/cm2 in complete expansion media (CEM) consisting of Iscove modified Dulbecco medium supplemented with 9% FCS, 9% HS, 100 U/mL penicillin, streptomycin100 mg/m , and 12 mM L-glutamine
Good Luck
I recommend checking everything Daniil said, making sure your T-75 is not low adherence. In addition, any chance your centrifugation conditions are different?
Hello. The primary cultures are very delicate. I guide you not do more than two passages, as it may suffer some differentiation process naturally. In every experiment, I perform a different pool of cultures with their own controls, so I will always have new and fully functioning cells.
Don't change the flask. Start and continue on the largest. Make a pool of cells of 2 animals, and count 2X10 ^ 4 cells per flask.