THP-1 culture problem
Posted 24 January 2011 - 08:00 AM
I've cultured THP-1 some years ago using RPMI 1640 from GIBCO supplemented with 10% FBS and 1% pen-strep and had no problems. Recently we acquired THP-1 from ATCC and a colleague seeded the cells using the ATCC special RPMI 1640 medium and prepared stock vials that were sent to me. I've seeded one of those vials using the RPMI 1640 from GIBCO and not from ATCC and I'm having a little problem: the cells are alive, but not multiplying.
I seeded the cells with a cell density of 2x10^5cells/ml. Since the cells were not growing, I decided to increase the cell density but the problem continues. The cells look great and healthy, but the only thing is that they are taking too long to multiply but they are wasting the medium which means that every 2/3 days I need to replace the medium and I think that each time I centrifuge the cells to replace medium, they slow down their growth rate again...
They are now in a T25, upright, resting in fresh medium since yesterday...
I compared the medium composition from ATCC and GIBCO and the main difference is in glucose (4.5g/L vs 2g/L, respectively) and sodium pyruvate(0.11g/mL vs 0g/L, respectively.
Does any of have had this problem before? Any tip?
Thanks a lot in advance!
Posted 27 December 2011 - 12:49 AM
I have also difficulties with culturing THP-1 cells: I grow them in RPMI supplemented with FBS, Pen/Strep, Sodium Pyruvate, Glucose, L-glutamine and non essential amino acids. Since they were not growing at the rate they're meant to be, I was advised to add a vitamin complex to the medium. But since then, they began to die very fastly and i re-replaced the medium of some of the cells. Neither cells without vitamin nor with vitamin are recovering. Any ideas?
Thanks a lot!
Posted 09 February 2012 - 04:14 PM
I've cultured THP-1 cells with my last job. When you bring them up from nitrogen they do take almost a week to start multiplying so if you are splitting them within 3 days of thawing them or changing the media then this maybe one of the reasons why they are dying. Also, they are much happier with a density of 3x10^5 - 5x10^5 cells/ml. You should add about 1mL of extra FBS in your T25 flask when you bring them up to facilitate their growth. Leaving the T25 to stand is also a great way to help them along as they prefer cell-cell contact. Be sure to also count the viability with trypan blue when you first bring them up as well.... if you have about 30% dead the chances of growing are slim. Hope this helps and good luck.