I recently started working with THP-1 cells as an alternative to MDMs, and have been having difficulty growing my culture. We are trying to get them to a point where I can try a couple of differentiation protocols and isolate RNA. According to the protocol we got from collaborators, this requires 10x10^6 cells per 10cM plate. This seems like a huge amount, and, given that these cells do not like over-crowded conditions, I am confused as to how I can grow up this many cells.
My main question is, how long does it typically take to grow up these cells, and how many cells are needed for differentiation for RNA?
I thawed the cells two weeks ago, and have been seeding them at 3.5x10^5 and 450,000x10^5 in T75 flasks in RPMI+10% FBS+0.05mM BME+1mM sodium pyruvate+1%HEPES. My last passage, I had 11 million cells total from 10 T75 flasks, which is still not enough. Is there a trick I am missing to grow these cells? It seems unreasonable to have to use more than 10 flasks just for one experiment. My current conditions are 5X10^5 cells in 10mL media in upright T75 flasks. The cells are on passage 8 now.
This is our first time working with non-adherent cells, so we really don't know if there is something wrong with the cells we thawed and how the process is supposed to go. Any help would be greatly appreciated!
Hi Emily,
In my hands THP-1 cells are somewhat slow growing (double every ~36h) but otherwise healthy. Your seeding densities are very low and about 1/10th of what I use. I agree you don’t want these to get overgrown but in my hands they are happiest at a density of 5x10^5/mL to 1x10^6/mL though you want to be careful to not exceed the upper limit. The ATCC seeding recommendations are slightly lower than mine at 2-4x10^5/mL. Like you I grow these in T75 flasks stood upright, in 10-20mL of media. At the upper end of this, the density for seeding your 10cm dish is readily achievable with 10mL media or if sticking with the lower ATCC values ~20mL. Below 5x10^5 per mL they are noticably more slow growing.
For harvesting for RNA, I would usually use 2 x 10^6 cells of material for a kit based extraction e.g. RNeasy. The only other thing to bear in mind is that assuming you are differentiating to macrophage or DC the cells will stop dividing so what your cell number is when you start differentiation will be more or less what you finish with.
Best
Ed
Typically when i grow these cells, they have 1-2 week lag after defrosting. After that they are usually pretty good natured cell line. I agree that the cells are underseeded and that will hinder their growth. Until they are fully recovered from defrosting you can do a half medium change every 2-3 days. In a non adherent culture, the viable cells should look round and shiny and you should see more of them with proliferation.
I think your issue stems from not having worked with a suspension cell line,
with these cells the seeding density is much higher as they are in suspension an not confined to the surface of a culture vessel. for example, i seed raw264.7 (adherent macrophages) at 2.1x10^6 per t75 but for thp-1 i seed at 6x10^6 (0.3x10^6 per ml).
I seed THP-1 in t75's at 3x10^5 per ml in approximately 20ml of RPMI until they reach 1x10^6 which usually takes 3-4 days.
are you seeding t75 flaks at 5x10^5 per ml or is that the total density per flask? if it is the latter, then the cells will struggle to grow to a reasonable density. when you seed too low there are not enough growth factors (secreted by the cells) in the culture media. this is also a good reason to not centrifuge your cells every time you subculture as you lose the growth factors and any other secreted molecules.
i would be inclined to check the viability of your cells (with trypan blue exclusion assay) as they have clearly not been growing correctly.
I agree that the cells should be kept at a density between 3x10^5 and 1x10^6. After thawing, they grow very slowly for a couple of weeks. I would also just feed the cells as needed instead of replacing all of the media until you have the number of cells you need. Passage 8 is getting to the upper limit for cells you want to differentiate.
for definite their concentration should be more than 1x10^6 to grow perfectly.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line