Hi. I'm currently culturing THP-1 (ATCC TIB-202) cells for my experiment and have been experiencing persistent clumping issues. I would greatly appreciate any advice or recommendations you could provide.
Media Composition:
450ml RPMI 1640
50ml FBS (not heat-inactivated)
5ml L-glu
5ml P-S
50ul 2-mercaptoethanol before use
Seeding Concentration:
I understand that a seed density for 3-4E5 cells/mL is recommended. However, most of my lab members maintain a higher density, as they are also suffering the same issue - typically more than 8E5 cells/mL. Our usual maintenance protocol is:
- 8E5 cells/mL (at this concentration, the cells stop to clump)
- 100 mm dish
- 10mL total volume
- Subcultured every 2-3 days
Subculture Procedure
1. Collect the cells into a centrifuge tube
2. Centrifuge at 1500rpm for 5 mins
3. Discard the supernatant and resuspend the pellet in fresh complete meidum
4. Aliquot 10mL of the resuspended cells into a new 100 mm dish
The attached images were taken today (2 days after subculture from 4E5 cells/mL concentration). I'm currently growing two separated thawed vials (refered as A & B), both of which had been stored in liquid nitrogen since last year.
Thank you in advance for your kind assistance :)
Hello Jeongmin Lee
THP-1 cells naturally tend to clump together, particularly when grown at high densities. Therefore, the recommended seeding density is between 3-7 x 10^5 cells/ml.
There are other reasons for clumping.
a. When cells are overcrowded or when media isn't changed regularly, there is depletion of nutrients and buildup of waste products which can trigger clumping. So, make sure that you are using fresh, high-quality media. Please note that THP-1 cells grow better in conditioned media. Therefore, whenever you passage cells, leave 1-2ml of old media and add fresh media to the culture. Remove dead cells as they can cause clumping. DNA released from dead cells tend to stick to other cells and debris causing formation of clumps.
b. As THP-1 cells divide, the new cells may remain attached to the parent cell, contributing to clumping. So, when passaging, gently triturate the clumps to break them.
c. Adding 2-mercaptoethanol to the medium may help prevent clumping. Add 2-mercaptoethanol to a final concentration of 0.05 mM in culture medium.
d. I suppose you have just thawed the cells recently, then you need to increase the FBS content to 20% for the first two to three passages, then you can switch over to 10% FBS for successive passaging.
Good Luck!
Hi there,
- THP-1 cells naturally grow in clusters; some clumping is expected, but excessive clumps can signal stress or density issues.
- Stick to recommended seeding (~3–4×10^5/mL) — higher densities (>8×10^5/mL) often force aggregation.
- Gentle pipetting during subculture helps break up clumps without damaging cells.
- For more uniform suspension cultures, substrates like DendroTEK dPGA | Protease-Resistant Culture Matrix can reduce aggregation and improve consistency.
Source: I work at DendroTEK Biosciences. www.dendrotek.ca
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