HepG2 are adherent, epithelial-like cells growing as monolayers and in small aggregates, have a model chromosome number of 55. HepG2 cell line was derived from the liver tissue of fifteen year old male with differentiated hepatocellular carcinoma. Not tumorigenic in immunocompromised mice. Secrete plasma proteins, such as albumin, transferrin, fibrinogen, a-2-macroglobulin, plasminogen. Cells respond to stimulation with HGH.
follow this protocol....
1. Use Eagle's Minimum Essential Medium (DMEM and RPMI1640 also works well) supplemented with 10% FBS. Discard culture medium every 2-3 days
2. To passage cells, briefly rinse cell monolayer with 1xPBS twice and add pre-warmed (37°C) 0.05% Trypsin-EDTA solution for 5 - 7 minutes
3. Once cell layer is dispersed (5-7 min at 37°C) deactivate Trypsin by adding equal volume of complete growth medium with 10% FBS
4. To avoid clumping don't agitate the cells by shaking the flask while waiting for the cells to detach
5. Split cells 1:4 every 3 days (recommended), or 1:8 every 6 days.
6. Cultures should be incubated at 37°C in humidified atmoshere with 5% CO2
Note- HepG2 cells have been demonstrated to be Neomycin G418 resistant (400 µg/mL).
This depends on your experimental requirement. If u want to just maintain cells you can seed around 1 million cells/t25 and split the cells after 48-72 hrs in 1:4 changing the medium every 24 hr. The number of cells in t25 flask is around 3 million and 1 million/well of 6 well plate. Accordingly u can calculate for diff well formats. please remember the doubling time is around 24 hr. if u really want to be precise just make a growth curve under your culture condition. The seeding density should be kept such that the cells should be 50-60 % confluent for microscopy expt and 80% confluent for transfection study.
hi I am working hepg2 cells and I need to know do I need collagen coated flask and plates or normal polyesterene and nonpyrogenic will work
You do not need collagen coated flasks, they grow fine in using polyesterene
I am working with HepG2 cells. Initially when I deforst the vial the cells were doing really well. Now they are growing very slow. I am using DMEM low glucose media, 10%FBS, antibiotics and Non-essential amino acids. I am using t25cm flask for culturing. color of media remains same too. confluency rate is so low even after 48 hours
Please guide what can be the possibilities.
Dear Sadaf Moeez ,
Try to use T75 cm flask for culturing . HepG2 is growing at 37°C, 5%CO2 in a complete medium consist of RPMI1640 instead of DMEM supplemented with 10% FBS . These cells grow well in RPMI.
FBS should be inactivated while preparing medium.
Abdulelah Ahmed Thabet CAN YOU TELL ME ABOUT THE COMPLETE MEDIUM.. THANKS
Yes you can add antibiotics for sub culturing .
Complete media consisted of: RPMI1640 media supplemented with 10% v/v FBS + 1% antibiotics (100 U/ml Penicillin/Streptomycin)
http://www.hepg2.com/
Dear Sadaf Moeez the answer of your Q "do I need to add antibiotics for sub culturing or not?" is in the attached links.
https://www.researchgate.net/post/Do_you_routinely_use_antibiotics_in_culture_media_What_do_you_use
https://www.thermofisher.com/eg/en/home/references/gibco-cell-culture-basics/transfection-basics/factors-influencing-transfection-efficiency.html
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