I initiated LCLs 8 weeks ago with 1ml EBV/flask and RPMI with 20%FBS. We are facing the following problems.
1.The cells are small in size ,not expanding as frequently as suggested by established protocols.
2.Cells are being transformed, but the size of the rosette is not getting bigger.
3.We have cells attached to the bottom of the flask.
Hello Akhila Sankar
Did you add phytohemagglutinin (PHA) during transformation? Also, during culturing, you need to add 2mM L-glutamine to the culture medium and try to reduce the percent FBS from 20% to 15%. Preferably, use FBS which has not been heat-inactivated. The culture should be fed in three to four days depending on how fast the cells grow. In this case, you try to remove half the volume of spent medium and replace with equal volume of fresh medium after the cells have settled to the bottom of the flask. Also, the growth of cells can be affected if the surface to air ratio is not properly maintained in the culture flask. So, use proper volume of culture medium in the culture flask. Lymphoblastoid cell line grow as suspension culture, and they have a tendency of form clumps.
I hope this helps.
Best.
Malcolm Nobre
Thankyou for the suggestions.
1.We have not stimulated the cells with PHA but L-Glutamine was already present in our media.
2. FBS is not heat inactivated. But, we are continuously using 20% FBS throughout our LCL transformation.
3.The growth of cells are very slow, even after feeding them with fresh media on every 4th day, and slowly scaling up with increased volume from 2mL.
4. Our main concern is:
We have initiated a culture in on 5/5/21 in vented T25. We have seeded the culture with approximately 5 million cells, but as of today 5/7/2021,observed clumps of smaller size. Since the clumps are not getting bigger ,we have sub cultured it only once.
- Badges
- Science topic