I'm currently conducting myeloma cell (MM1s) culture for experimentation. I'm using RPMI-1640 medium with 10% FBS and 10% penicillin+streptomycin. I seed 5*10^6 cells in a 150mm petri dish with 20ml of medium and maintain them every three days. However, with each culture, the cell number doesn't increase significantly, and viability fluctuates between 40-60%. After just a couple of maintenance cycles, all cells die. I need a large quantity of cells for my experiments, so I'm trying to scale up the culture while maintaining and conducting experiments simultaneously. However, I'm stuck because I can't perform experiments, and all the cells are dying. What could be the problem? Here's my maintenance protocol:
Even with this method, both viability and cell numbers are very low. I reduced the centrifuge speed because lower rpm CFG was said to aid in dead cell separation. Previously, I centrifuged at 1800rpm for 3 minutes, but when I changed to lower rpm, viability increased by about 20% (approximately 60%). The pellet appears visible. When I observe the cells under a microscope during culture, they don't seem to be in good condition. Changing the media doesn't yield different results. What could be the problem?
Hello Iris Yoon,
I have some suggestions which you could incorporate in your protocol.
1. Subculture cells before they reach confluence because cells in over-confluent cultures begin to form rosettes with necrotic centers.
2. Use a subcultivation ratio of 1:2 to 1:4.
3. MM1S is a semi-adherent culture. So, you may have to gently scrap the loosely attached cells with the help of a cell scrapper to get all the cells in suspension.
4. In your maintenance protocol, why do you resuspend the cell pellet in PBS? Use complete growth media to resuspend the cell pellet.
5. You should supplement your growth media (RPMI + 10% FBS) with 2mM L-glutamine.
Hope these suggestions work!
Best.