I'm trying to subculture NHBE cells from Lonza, however, the cells seem to not adhere to the new flasks after detachment. I've followed Lonza detachment protocol (2 mL/25 cm2 of Trypsin 0.05% and EDTA 0.02% for 3 to 4 minutes) and there is little to no cell death post this procedure. I've seeded the cells into flasks of the same brand (TPP) that they were cultivated following the same conditions (without collagen coating), and some cells seem to adhere short after seeding. However, a lot of cells do not adhere, despite being alive, eventually dying. The cells that adhered began to detach and die in the following days. Does anyone know how can I solve this problem?
I don´t know the particular cell line, but from general cell culture I could suggest a few things. First, trypsinization does not seem to be hard, 0.05% for 3 or 4 minutes shouldn’t damage cells so much. When you say there is little death, how do you study it? Tripan blue sometimes is not sensible enough, if able, you could try Propidium Iodide or 7AAD. You should see round cells with clear borders after trypsinization. Do you have a centrifugation step after trypsinization? Maybe you have a problem there, check that centrifugation speed is about 300 g. Also check if your serum concentrations are the correct ones on the growth medium after passage.
If that is not your problem you could perform a serum coating to help cells attach.
Hope it helps!
Diego
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