I have been culturing HepG2 for carrying out some in vitro studies. The cells were maintained in MEM (Gibco) supplemented with 10 % FBS. 1x Ab/Am was also added in the growth medium. Cells were found to clump extensively. Care were taken to break the clumps by vigorous pipetting during passages, yet the clumps were developed by the following day. Growth rate was also found to be low with population doubling time of 10 days. Anybody with hands on experience may throw light into this puzzling observation.
I'm not sure about HepG2 specificially (although my coworkers are now starting to grow them), but in general also consider whether you are using the proper volume of media- too much or too little can cause problems- stunted growth and clumping are possible. Are you using an indicator such as phenol red? Monitoring the pH might be useful. Also are you using trypsin to dissociate or something else? Perhaps they are clumping because of your dissociation method and you can't remove the clumps later on? Also make sure you are using the recommended number of cells when you prepare fresh flasks. Do you check them using trypan blue, etc to see if they are healthy? These are just some random things I can think of to maybe consider if you haven't already. There are so many variables!
Hi Prajith,
If you are using Trypsin solution to detach the cells from the plate during splitting procedure make sure it contains EDTA.
EDTA will help reducing cell clumps significantly.
Best of luck!
@ Dr. Andrea, Thank you for those invaluable inputs.
@ Ms. Naneki and Dr. Milica, Thank you for your suggestions. I've been using Trypsin-EDTA solution (0.25 % Tryspsin and 0.28 % EDTA, in PBS, w/V) for detaching the cells. I hope that is fine.
@ Ms. Naneki, 6.5 mL media per T25 flask were used in the culture. Media have phenol red indicator and the media were neutral. I used to take an effort to break the clumps during the passages by means of gentle thrusts of the cells with media against the bottom of flask using pipette. I'm not sure whether it has some adverse effects on the growth of the cells. I used to give 1:2 splits from approximately 80% confluence flasks and routine cell viability tests using trypan blue assay during subcultures have shown few dead cells that were removed by discarding the supernatant media to the maximum extent possible on every occasion of attending flasks.
Rectifications in the techniques are invited if there are.
In my hands, HepG2 grow faster in DMEM+ 10% FBS than in MEM+10% FBS.
Clamps are normal and very difficult to dissociate, even when using trypsin+EDTA and rigorous pippeting. In the ATCC website, HepG2 also seem to appear to grow on clumps.
With regard to the slow growth, this sounds indeed problematic, maybe it is because of mycoplasma.
- Badges
- Science topic
- Similar topics
- Engineering
- Materials Engineering
- Powders