I am new to culturing HepG2 cells and they are currently cultured in DMEM with 10% FBS and pen/strep. Each time I passage them I am wash with PBS and then incubate with TrypLE for 8 minutes. I then neutralize the trypLE, spin down, and resuspend the cell pellet by pipetting up and down. I also try to passage them every 4 days and keep them below 80% confluency.
Is there a better method to culturing them or am I doing something in particular to cause the clumps to form? I've been culturing them for about a month or two now and it seems like they are forming more and more clumps the more I passage them. These clumps are also visible by the naked eye in the T75 flask I use as well.
Please let me know if you have any suggestions for fixing my current methods!
Hello Teagan Dean,
HepG2 cells, when they reach confluency (around 70% and above), tend to form 3D structures and clumps. To avoid excessive clumping, it is recommended to subculture HepG2 cells before they reach 70% confluency, with 60% being a good target. This ensures that the cells are still relatively dispersed when they are detached and replated, reducing the chances of clumping.
Moreover, during subculturing, gentle trypsinization is crucial to avoid cell damage and clumping. Ensure proper trypsinization time as over digestion with trypsin can lead to clumping because DNA released from dead cells is sticky and can cause cell clumping by binding to other cells and debris in the surrounding environment. You should also avoid excessive agitation.
Best,
Hi Teagan:
Sorry to hear you are having this issue. Clumping is quite common across cell types in vitro.
- HepG2s are semi-adherent and naturally form clusters; some clumping is expected.
- Try shortening TrypLE incubation (8 min is long) and gently triturate to get a cleaner single-cell suspension.
- Avoid spinning too hard — 1000 rpm can compact them; lighter centrifugation helps.
- For stronger attachment and reduced clumping, DendroTEK dPGA | Protease-Resistant Culture Matrix provides a stable surface compared to standard coatings.
Source: I work at DendroTEK Biosciences. www.dendrotek.ca
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