I am trying to culture human preadipocytes. After receiving a cryopreserved vial of preadipocytes, I cultured the cells in DMEF/F-12 media containing 10% FBS, 1% ultra-glutamine, and primocin. Even after 48 hours of culture, most of the cells are still floating and only about 10% are attached to the surface. Did anyone here have a similar problem? Do you have any suggestions for me?
Hello Subodh Sharma
I would like to suggest the following.
Sometimes delicate cells have to be handled with care. When you thaw the cells do not dilute and centrifuge the cells since these actions may be more harmful to the cells than the effect of residual DMSO in the culture.
You may try to culture the cells in poly-L-lysine-coated culture vessel to promote cell attachment. Prepare a poly-L-lysine-coated culture vessel (T-75 flask at 2 μg/cm^2 is recommended). To obtain a 2 μg/cm^2 poly-L-lysine-coated culture vessel, add 10 ml of sterile water to a T-75 flask and then add 15 μl of poly-L-lysine stock solution (10 mg/ml).
Leave the culture vessel in a 37 degree C incubator overnight (or for a minimum of one hour). Rinse the poly-L-lysine-coated vessel twice with sterile water and then add 20 ml of complete growth medium. Leave the vessel in the sterile field and then you proceed to thaw the cryopreserved human preadipocytes cells.
Some points you need to know.
1. Refresh the culture medium the next morning after establishing a culture from the cryopreserved cells.
2. Change the medium every three days, until the culture is approximately 70% confluent.
3. Once the culture reaches 70% confluency, change medium every other day until the culture is approximately 90% confluent.
4. Subculture when the culture reaches 90% confluency.
Hope this will help!
Best.
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