We are working with Small Cell Lung Cancer cell lines, H209, H146, H69. These cells usually grow in floating clumps. Recently we are having problems maintaining their viability. After a few days the cells look small, with many vesicles and then die. The cultures are clean from mycoplasma and other contaminants. Culture medium is RPMI, Glutamine, 10%FBS, antibiotics.
ANy suggestions would be of great help.
Thanks
I have never worked with any of those established SCLC cell lines before, but I worked with our own patient-derived SCLC cell cultures from our hospital with varying growth rates. I cannot comment much on the information you provided, but I suspect that there may be something less desirable with the way you subculture and the subcultivation ratio. Also, depending on the type of SCLC you are culturing, the cells might grow slowly. My suggestion is to gently disperse your culture during subculturing and split it into fewer flasks (e.g., 1 to 2). I usually use serological pipette pipetting-in the cell suspension, pointing towards the bottom of T-75 flask, and then pipette out the suspension, using shear force to disperse the aggregates. You can repeat this several times until you have smaller aggregates. Never let your cell aggregate to form a necrotic core in the middle. Pay special attention also when you thaw the SCLC cell line from a new liquid nitrogen stock as they are more sensitive to the thawing procedure and DMSO in freezing medium. I normally use fast-thawing at 37 degree C and then transfer 1 mL of cell suspension to 3 mL of FBS dropwise.
Hope this helps!
Thank you Win for your detailed suggestions. The gentle dispersion idea is a good one. We are currently using light centrifugation to isolate the aggregates. You are right regarding the necrotic center of large aggregates.
Thanks again,
Michael
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