HepG2 cells tend to grow in clusters, often forming aggregated 3D structures.

Don't let your cells to over grow, they will form clusters than monolayer if you wait too long. Harvest them ~ 70% confluence with help of trypsin. wash cells with warm PBC/incomplete media and break pellet very gently to disperse cells into single cell suspension. I use 5ml syringe and 18 gauge needle for this. and it works fine for me.

Also don't seed too low or too high number of cells, it affects their growth and morphology.

I hope it will help.

Hi Mamta

I too have been working on HepG2 cells in the same media composition. I trypsinise a 10 cm culture dish with 1 ml of trypsin for maximum 3 minutes when they are 70% confluent. To inactivate the trypsin I add three times of trypsin, complete media (3 ml) and pipette in and out quite well to break the clusters of cells. And after centrifuging again I do the same to resuspend the cells for plating. And it seems to work fine. 

The important steps here are the trypsinisation time and the formation of single cell suspensions. You can check it under a microscope before plating to confirm.

Hope this helps.

Hi Mamta,

I also work wit HepG2 cells, I used DMEM medium supplemented with 10% FBS, 1mM sodium pyruvate and penstrep antibiotics.I trypsinise for 3 min then centrifuge, wash the cell pellet with medium and centrifuge again, after that just resuspend to disperse the pellet in medium and plate to the dish. it's work well to me.

Hope it works.

Hello

you can use 0.05% trypsin +0.02_0.05% EDTA for 5-7min it's better to warm the medium to 37C HepG2 cells tend to aggregate and pile in many type of medium (e.g. DMEM), but behave very well ( confluent monolayers, no aggregation) if you grow them in Hams F12 medium (with 5-10% FBS)

use pre-warmed 0.5 % trypsin and 0.2% EDTA solution in DPBS for approximately 7 - 10 minutes for better result. I suggested you for using pre-warmed trypsin EDTA solution for establishing the optimum temperature so that you can minimize the cell death

Hope help you , Good Luck

http://www.hepg2.com/

https://www.dsmz.de/catalogues/details/culture/ACC-180.html?tx_dsmzresources_pi5%255BreturnPid%255D=192

Hi Mamta,

While day-by-day working with HepG2 cells for more than a decade, I’ve learnt to strictly obey the following protocol about their tripsinization procedure (to your very matter of quation, please pay special attention to the points ##3-5):

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.

Cultures should be incubated at 37°C in humidified atmosphere with 5% CO2

Best wishes,

Ilya

Thankyou so much to everyone for the very usefull answers.. I will definitely use this. 

Hello mamta,

We also working on cell culture since 3 years experienced various problems on protocol of different cell lines. In this case Dr. Ilya Tsyrlov gave superb ans.  i would just like to add one thing, in step 3 add more amount of media to deactivate trypsin and immediately centrifuge the cell suspension. Remove the superntant media and disperse cells gently, split it as per point 5.

Hope it will work.

Dear Mamta,

I had worked in  HepG2 cells for more than one year , I was act on cells after 6 days incubation so cells  was highly attached with colony (Clumps) .

 So to obtain a single separated cells was so hard, only  the  following protocol about their tripsinization procedure was the most effective one :

(( All the following steps suitable  for  flask 75 cm2 & media volume 15 ml))

1) Use  Essential Medium  supplemented with 10% FBS.(15 mi)

2) Discard culture medium , briefly rinse cell monolayer with 1xPBS twice (each one by around 10 ml)

3) Add pre-warmed (37°C) 0.05% Trypsin-EDTA solution for 5 – 7 minutes (Do not Shaking to avoid cluster formation or cell aggregation).

4) Once cell layer is dispersed (5-7 min at 37°C)- some times may incubate up to 10 min.- deactivate Trypsin by adding equal volume ( 10 ml ) of complete growth medium with 10% FBS.

5) Centrifuged for 3 min in 1200rpm

6) Discard the supernatant & resuspend  the pellet in 1 ml (medium with 10% FBS).Then  count by using T.B.

7) Take the required volume from the upper layer of cell suspend , to avoid any clumps.Then inje3cted in media, mix well. Finally  incubated at 37°C in humidified atmosphere with 5% CO2

Dear Mamta

I am working in Hepa-G2 cells,so I agree and confirm the same protocol and trypsinization procedure of Ghada Al toukhy and making sure that the 1xPBS for rinse cell and growth medium are pre-warmed (37°C).

Dear All

I need to move the Hepa-G2 cells from one place to another during the summer where the temperature is higher than 37 ° C, distance 400 kilometers and it takes approximately 6-8 hours to transfer them to the laboratory.

What should I do to protect the Hepa-G2 cells (keep them healthy)?

I THINK YOU HAVE CHOOSE. TO FREEZE THE CELLS INCRYOVIAL AND MOVE IT  IN FRIDGE AT -80 C. ANOTHER WAY TO CULTURE CELLS INTO FLASK AND MOVE IT IN AN INCUBATOR AT 37CO WITH ATMOSPHERE OF 5% CO2.

Dear Mamta,

To trypsinize HepG2 cells, you need to wash the cells monolayer with pre-warmed PBS solution (1x) twice. Then, use pre-warmed trypsin containing 0.02% EDTA for 7min. Try not to hit the flask because they will aggregate. Then, after incubation time has passed add the required amount of media (double the amount of trypsin you used). Its better to use EMEM media at first 2 weeks and then switch to DEMEM media gradually. You can continue with EMEM media. Before seeding the cells, try to aspirate the cells up and down with micropipette to remove the aggregation.

Hope this help , Good Luck with them!

HepG2 cells use to make clumps instead of growing in a monolayer. For some of my experiments, I need to have their monolayer e.g for scratch assay. I used collagen to coat the plate before seeding the cells, and then I got the monolayer. If coating with collagen is not disturbing your experiments, this will also help to see the normal morphology of the cells and to get their monolayer.