For the past few weeks, I've been attempting to passage U-87-MG cells in our lab. However, after thawing, instead of reaching 80% confluency within 24 hours, they're taking 3-4 days to achieve the same level of growth.
Also, upon seeding in a 12-well plate these cells are failing to adhere to the plate surface, leading to the loss of cells.
NOTE- We ensure that all chemicals are freshly prepared, and we use the suitable media for U-87-MG cells. Moreover, our other cell lines are exhibiting normal growth without any issues.
Hello Bhaskar Chatterjee
I feel you need to revisit your freezing protocol. Have you frozen U-87-MG cells yourself or has somebody else prepared the frozen stock?
For good results, you should harvest the cells during their maximum growth phase (or log phase) and should have greater than 80% confluency before freezing. Also, it is important to consider the optimal concentration for freezing cells which may vary depending on cell type. Typically, the concentration of cells in the cryogenic vial should be within a general range of 1x10^5 - 1x10^6 cells/ml.
Additionally, when you freeze cells, you should follow the standard protocol of freezing namely, gradual freezing at -1 degree C/min using Mr. Frosty freezing container.
What was the cell viability on thawing?
Best.
The slow growth and poor adherence of U-87-MG cells could be due to several factors. Here are some potential reasons and solutions for the issues you are encountering: Thawing and passaging procedures can affect cell viability. Ensure that the U-87-MG cells are properly thawed, and the viability is assessed using a trypan blue exclusion assay or other viability assays. Consider obtaining a new vial of U-87-MG cells from a reliable source to rule out issues with the cell quality. Even though suitable media are being used, there may be variations in the culture conditions that affect U-87-MG cells differently than other cell lines. Double-check the pH, osmolality, and the presence of necessary growth factors in the media. Additionally, confirm that the CO2 concentration and temperature in the incubator are appropriate for U-87-MG cells. Consider optimizing the trypsinization time and concentration for U-87-MG cells. Some cell lines, including U-87-MG, may require a specific substrate coating for proper adhesion. Consider using poly-L-lysine or other suitable coatings to promote adherence.
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