Hello everyone,
I am currently growing CCD33Co cell line that I bought from ATCC. The media I am using is EMEM (ATCC cat#30-2003) mixed with 10% FBS and 1% strep/penicillin. The cells were plated on a 10 cm plate. The cells are growing very slowly since I took them out of the ATCC vial. It has been 3 weeks and they are still at about 40% confluency. The cells are not quickly dying to say that they are dying than growing (I am keeping tabs on floating cells). My colleagues have seen the cells and say they are fine, meaning they are not contaminated or anything obvious. I am not sure how to approach this problem because I am very new to cell culture techniques and growing cells.
Does anyone have any suggestions?
Thank you!
Hello Tara Mahmood
How did you thaw the cells?
Since you are new to cell culture, I would like to suggest the following.
Quick thawing is necessary in order to maintain high viability upon thawing.
I am listing the steps of the thawing protocol.
1. Remove the frozen vial from the liquid N2 storage.
2. Loosen the cap of the vial slightly to release the pressure inside.
3. Thaw the cells in a 37ºC water bath.
4. Keep the lid of the freezing vial above the surface of the water to lessen the chances of contamination.
5. When the cells are almost thawed (only a small piece of ice) move the vial to the tissue culture hood.
6. Wipe the outside of the vial with 70% ethanol and remove the top.
7. Carefully remove the cell suspension using a sterile Pasteur pipette.
8. Transfer the contents to a centrifuge tube containing 9 ml of appropriate pre-warmed complete culture medium.
Now, there are two schools of thought on whether the cryoprotectant should be immediately removed from the cells:
1. In some labs, the cells are centrifuged at 200-300g for 5-10 mins, and the cell pellet is resuspended in fresh cell growth medium prior to placing cells in a culture dish.
2. However, some cells are extremely fragile after thawing and that centrifugation may increase cell death. So, it is recommended that the cells be plated, and that the growth medium be changed after the cells have attached (7-8 hours) or the next day.
You will have to decide which one of the above method will work well for your cells.
Another important point you need to note is that after thawing, the cells have to be plated in a smaller flask like T-25cm^2 flask and not in 10cm plate.
Hope this information will help!
Best.
Malcolm Nobre Thank you for the instructions. I did follow a similar protocol to thaw out the cells.
I did talk to ATCC and it turned out they sold me the cell line at passage 24 and the max passage this cell line will go is 29 before the cells become abnormal.
ATCC thinks that maybe the cell line given to me was not as viable as they thought.
My boss wants me to move onto another cell line.
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Virology
- Virus