Hello,
I am working with primary cell cultures of pig buccal fascia and mucosa. I usually culture them for 7, 14 and 21 days and freeze them with 10% DMSO on each time point. I have frozen tubes from 2022 and 2023. The point is, after thawing the samples, ADAM cell counter shows, that in both cases, approx. 90% of cells are viable. Then, I am seeding both samples on culture plates, with the same media, according to instructions*.
I have tried it multiple times, and the pattern is always the same: most of the 2023 cells adhere and grow nicely, but 2022 cells do not attach to the plates, they float.
I considered the fact that they might have been damaged while being in -80C, but if that was the case, then I believe the cell counter would show low cell viability. But each time viability is oscillating around 75-90%, for both 2022 and 2023 cells. And I only use the number of viable cells while seeding.
I am mixing my own media (DMEM with added L-glutamine, FBS and antibiotic-antimycotic mix), the incubator has 37C, 5% CO2. The cells are in the 15 mL tube under the hood, while I am counting the cells, but it takes around 5mins, probably less.
I tried increasing the percentage of FBS. It helped somehow, but not much. Most of the cells are round and still floating.
If you have any suggestions for such a peculiar problem, please let me know as soon as possible.
* https://www.thermofisher.com/pl/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/cell-culture-useful-numbers.html. I know that for thawed cells its better to use higher density so I do this (instead of seeding 300 000 cells on 1 well in 6 well plate, I seed around 320 000).
Hello,
Please consider treating your plastics with polyLysine or gelatin. It might help.
P.S.: Some of the cells are definitely degrading over time is stored on -80 instead of -150 or liquid nitrogen tank.
- Badges
- Science topic
- Similar topics
- Organizational Psychology
- Training