I've not worked specifically with BEnd.3 cells, but I have seen something like that happen with other cell lines...

Is there a chance you have a mycoplasm or bacterial infection of any sort? Can you take a picture of the cells in culture and post them here?

Your question was not included all information. Were clumps seen on the day of seeding or 3 days laters after seeding?

If you see 3 days after seeding, it should be normal because you are doing 3D and it is the way supposed to be. Must use ulra low attachment flask or nunclon spera flasks to easily get 3D culture of cells.

If you are not planning to have 3D culture, you can simply trypsinize to have single cells suspension and then recultre again. Use Nunclon Delta flasks.

Here are some pictures taken today, after 7 days of seeding (seeded last Monday, 7/15). We have been seeding in the same flasks after adding trypsin. We have been leaving the trypsin for 5 minutes, and because the cells do not come easily after 5 minutes of trypsin, we have been scrubbing the cells.

Some clumping is observed on Day 1 of seeding, but it increases after a few days. About 50% of the cells seem normal, and 50% are in clumps.

Also, last week we had a suggestion to increase the amount of FBS in our media from 10% to 20%. We changed the media to 20% this past Friday (7/19), and now on Monday (7/22) the morphology is changed-- there are some spindles extending from some of the cells. However, the clumps are still present. As you can see in the pictures, there are two layers of cells.

Lastly, I mentioned we have been re-using flasks when seeding the cells. We now have 3 different flasks. The flask that was re-used 3 times has the most clumps, and the newest flask (re-used 0 times) has fewest clumps, but it still has a few clumps.

Thank you for your help!