Anyone mind to share their HepG2 cells morphology here? I am now getting different cell morphology (most probably due to my technique) and I keep getting clumpy cells and the borders between the cells are not really defined.
I attached the picture which I get from different passage, if anyone would like to comment which one looks closest to how it should be?
Hi Ain, when you do your passages, how long does the trypsinization step take? if it's a short period, then you'll get these clumps... also, you should split them when they are 80% confluent or so, more than that may also yield these clumps.
Hello, I think that cells in A, B and D are ok; maybe cells in C are growing bad.
https://www.phe-culturecollections.org.uk/products/celllines/generalcell/detail.jsp?refId=85011430&collection=ecacc_gc
Here you can find more informations about your cell line. Moreover, there is a picture of Hep G2
Good luck
Hello Ain,
HepG2 cells, even if hepatocarcinoma cells, do not grow too quickly. In my hands, it takes roughly 48 hours to achieve 70-80% confluency. Also as for their growth morphology, they start growing in islands. One thing with HepG2 is that they tend to clump, so as Terezinha said, keep an eye on the trypsinization time. I use 3-4min at 37degC. And resuspension prior to seeding needs to be slightly vigorous, since if kept around for sometime, these cells will clump in suspension. If you are seeding these cells on coverslips, do poke the coverslips in the end gently, to ensure their submergence. Hope this helps.
P.S - As Maurizio alreay pointed out, your cells in A, B and D look alright
Regards
Aparajita
Hello Terezinha, thanks for your kind reply. I usually aim for less than 5 minutes for trypsinisation and if the cells still attached, I will gently agitate the flask. How long is considered short period then?
Hi Maurizio, the link is really helpful! Thank you so much!
Hi Aparajita, thank you for the tips! I am just curious to how quickly you achieved 70-80% confluence in just 48 hours? Do you split 1:4? My cells usually need 4 days to achieve confluence if I split them 1:4, hmm. Sorry for all these very basic question and thank you for all your response :D
I agree with Dr. Forte's answer. Also, Dr. Lahree is correct that HepG2s grow in clumps no matter how you try to get them into single cell suspension. In fact, they grow better when they have other cells close by.
Do not split HepG2 too much, 1:3 – 1:4 should be fine.
As noted before, HepG2 cells like “companion cells” in culture and some clumping cannot be avoided upon trypsinization. However, you can greatly reduce clumping if you perform a second suspension-centrifugation cycle and pass the final suspension through a sterile nylon mesh. This way you will achieve a more uniform suspension and distribution of cells when seeding onto the plates.
Dear Ain,
I agree with the previous responses about the quality of cell in A, B and D. But I usually done trypsinization in 1 minute at 37°C trying to fully cover the floor of the flasks. Then I beat the flask heavily, add 8-9 ml of fresh complete medium and finally pass the suspension through a needle for trypsinization to obtain less aggregates. If you need to count cells I suggest to work as me while if you need to pass cells and mantain use 1:4 1:5 dilutions. without the passage with the needle.
One curiosity. How old are your cells?
have a good time, i have also same problem with my hepg2 cells. I call to campany which i bought my cells but they told me these cells morphology is as you see. They tend to make clamps. But i thrypsinized my cells for 5 minutes and the by a needle i make cells single and then seeded in flask and now they are in good gesture i think.
hi, after trypsinization and addition of complete media I keep my HepG2 cells in suspension for 10 mins. The clumps will precipitate down. Then I use the supernatent and centrifuge in a fresh tube. In this way I'm able to achieve single cells in HepG2 culture. But recently I have lost the triangular morphology, and my cells are rounded. Can anyone help me with this? Also should I proceed with my experiments in these rounded cells?
I think image 'A' looks best. I have experienced clumping but not such a change in morphology. My suggestion is to allow the trypsin to work for a long period of time (close to 10 minutes), then add fresh medium and pipette up and down vigorously to loosen clumps. Next, allow the visible clumps to settle for about two minutes, then transfer the supernatant carefully into another tube for centrifugation. After centrifugation, loosen up the pellet vigorously with medium once more. This worked for me
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