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C3HT101/2 Cell Line not attaching to T25 flask- nightmare!!!! - (May/12/2013 )

Hi all

I received 1ml of frozen C3HT101/2 cells from ATCC last week. I followed the instructions sent to me exactly, down to every last detail and now the cells are not attaching to the flasks and generally causing me massive problems!

Upon receiving the cells (in 5% DMSO in base media) I thawed them quickly (2mins) in a 37 degree water bath, I then washed the cells in 9ml media, span down gently for 6 minutes and resuspended in 1ml base media (ATCC told me to use: BME supplemented with 2mM L-Glut, 10% FBS and 1% S/P mix)

These cells are contact sensitive- very sensitive to post confluence inhibition of cell division and as such are seeded at an extremely low number. I was provided with 3.8x10^5 cells in 1mL, viability was expected to be around 89-93% and I was told a T25 seeding density of 3.0x10^3 viable cells/cm2 was required. Therefore I calculated 3.8x10^5 x 0.9 to factor in viability (slightly arbitrary value) which meant I should have around 342,000 viable cells sent to me. I then calculated the 3.0x10^3 x 25 (as there are 25 cm2 in a t25 flask) which gives a seeding density of 75,000 cells per flask. From the 342,000 (approx) viable cells I could then seed 4 T25 flasks each with 75,000 cells in them. I added 250 microlitres of cell suspension to 4.75ml of media and left in a 37 degree 5% Co2 incubator. It has now been about 3 days and the cells are not attaching to the flask or doing what they are supposed to do. This is the second time we have bought these cells, and I am completely at a loss to what is going wrong.
All cell culture flasks are corning, I have had ATCC check they are suitable- these cells are not supposed to require anything to grow on such as matrigel or gelatin etc.

I am culturing two other cell lines successfully ( BCL1 cells and HEK293 ) and the first time I purchased these cells I didn’t listen to anything ATCC said, I used DMEM for media, and seeded at about 300,000 cells in one single T25 flask and they grew and attached beautifully O/N. Unfortunately I then had problems with these cells post cryo-preservation and decided to purchase again and do exactly as was instructed- and I’m now having this nightmare.

Please help- any tips or instructions on what I could do would be great.
Sorry for the massive detail, this is my first post!


Mycoplasma ? But unlikely if you got them fresh...


It could be the FBS - different lots can alter cell behaviour quite a bit. Check with the ATCC which supplier of FBS they use (it is probably their own) and see if you can get some of their FBS.



Thanks for your responses

I don't think mycoplasma is the issue, getting them fresh from ATCC and they haven't had any other complaints about this batch.

I think the FBS might be a good call actually-! It is the one thing I didn't buy new, I have just been using the stuff in the -20 that everyone uses. Although my BCL1 cells and HEK293 cells are made from the same batch and they are ok

If anyone else has any experience in this kind of thing please let me know! The cells seem to be dividing in suspension but forming sort of aggregates. I am going to do a trypan blue assay today and keep them alive whilst ATCC get back to me.



If attachement is the issue, I'd recommend looking at a vessel with enhanced attachement propoerties like these:


I don't think FBS is the issue, most likely you used a flask that was not treated with some coating materials that came with the flasks. You may use AddexBio Coat, a coating material that helps cells attach to a flask. here is the link: Or else, just buy some new flasks that are cell culture treated. Best of luck.