Hi everyone. I have started working with HeLa cells recently. I used stocks from someone else who has left the lab and no longer available for answering questions.
Anyways, I harvest when my HeLa looks about >80% confluency (see picture), but I always get low cell count. For example, I got around 1386000 cells at 90% confluency from a 100mm dish which seems very wrong so I am confused. Whereas for HEK293T, I can easily get millions of cells at confluency on a 100 mm dish.
Here is the protocol I usually use for a 100 mm dish. All media prewarmed to 37C:
- Wash with 5ml PBS.
- Trypsinize with 3 ml Trypsin for 5 to 7 mins
- Deactivate with fresh media. Usually I can see visible clumps at this points (They are quite prone to forming clumps as I have observed). These are impossible to dissociate. When counting cells, I see quite a few clumps on the hemocytometer as well.
Also, I notice that when HeLa cells reach confluency, they continue to grow and squishing one another. Usually at that point they form even more clumps when trypsinized and I lost more cells. So does anyone know a good split ratio for Friday so that they don't grow too dense by Monday the next week? I have tried up to 1:15.
I have grown HeLas before (as well as 3T3 and HL-1 cardiomyocytes), 1:15 ratio of passage should last you more than a few days (https://www.ruf.rice.edu/~bioewhit/labs/bioe342/docs/cell%20passage.htm#:~:text=Cell%20passaging%20or%20splitting%20is,Materials), I remember doing 1:2 or 1:4 splits for the weekends to get confluency on Mondays. According to the table 2 in the Rice Uni link, 1:15 should last you a week before full confluency. A low confluency can give stunted growth phase as well, I found that it takes a while for anything below 25%ish confluence to grow. As per your clumping problem, I found that gently tapping on the flash/plate would help me get rid of the big clumps whilst I was checking the cells on the phase contrast microscope. You do have to be careful with the tapping force. How is your trypsin, are you using fresh trypsin with trypsin aliquots. Is it subject to freeze-thaw cycles? Trypsin is another key crucial element.
Hello Loc Ngo
You may use the following protocol for passaging HeLa cells that are ≥ 70% confluent in a 10 cm dish.
(1) Aspirate off the cell media.
(2) Wash the cells with 5 mL PBS.
(3) Aspirate off the PBS.
(4) Add 2mL of Trypsin/EDTA. This is just enough to cover the surface of the dish.
(5) Return cells to incubator for ~2 minutes.
(6) Add 8mL of cell media to the dish. The FBS quenches the trypsinization.
(7) Pipette media up and down, and squirt it around the surface of the dish to remove adherent cells from the surface.
(8) Move 10mL of cell suspension into a 15 mL Falcon conical tube.
(9) Centrifuge at 1500rpm for 10mins.
(10) Discard supernatant, tap pellet and add 10ml fresh culture media.
(11) Move the appropriate volume of cell suspension into new 10 cm dishes.
(12) Spread the cells evenly by rocking the dish/plate back and forth. Do not spread the cells by swirling the media in a circular motion. This may result in clumping of cells in the middle of the dish.
The important points to be noted while subculturing HeLa cells.
1) Subculture cells when they are 70-80% confluent. When you harvest HeLa cells when they are >80% confluency (sometimes at 90% confluency), dead cells are likely to be formed in culture. Dead cells release DNA in culture. Presence of free DNA (DNA is sticky in nature) in cell suspension attract cells which bind altogether forming clumps in culture.
2) Also, over exposure of cells to trypsin during trypsinization can lead to cell death. So expose cells to trypsin for not more than 3-4 mins.
3) You need to get single cell suspension that will help in giving you an accurate count using hemocytometer. If the cell clumps are small, you may pipette the cell suspension a few times gently up and down to form single cell suspension. If you still do not succeed, you may consider using DNase I at concentrations ranging from 20-100µg/ml to avoid formation of clumps. For each cell type, the working concentration must be determined individually.
The recommended splits for HeLa cells in a 10cm dish are:
1) 1:5 dilution of cells in a 10 cm dish: Put 2mL of cell suspension into a dish containing 8mL of culture media.
2) 1:10 dilution of cells in a 10 cm dish: Put 1mL of cell suspension into a dish containing 9mL of culture media.
I would not recommend 1:15 dilution as it would change the characteristics of the cells.
Best.
hello, I had a similar issue with cell clamping. i had to reduce the amount of trypsin volume or concentration for trypsinizing the cells. Here are some links that discusses such issues.
https://www.akadeum.com/blog/cell-clumping/
https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/mammalian-cell-culture/cell-clumping-troubleshooting
Thanks guys for the answers. I think I might have figured it out (haven't tested my suspicion yet). I think the issue is my trypsin digestion is too gentle. Apparently for HeLa cells, the working trypsin concentration recommended by ATCC is 0.25%, but I have been using 0.05% as I have always used with other cell lines. I will try this out and see if this improve the dissociation and reduce clumping.
Great, I checked my dissertation and it seems 0.25% trypsin was what I used too. I do remember the less dilute trypsin being problematic for me as well. If you use a relatively strong trypsin with gentle tapping to disassociate cells you don't have to subject the cells to too much stress. The whole thing shouldn't last more than 5-10 minutes from trypsin to FBS inactivation. Are you using double trypsin wash? I did cell viability assays with cell counters, was routinely getting >90-95% cell viability and confirmed with annexin + 7AAD/PI staining with flow cytometry.
Hi,
I would recommend washing in trypsin instead of PBS, gives a bit of a headstart...you may also consider switching from the dishes to other culture vessels (i.e. T75 flasks).
And yes, there is a difference between initial optical confluency and the timepoint when contact inhibition kicks in...so they grow (a bit) tighter, once they cover the full vessel area.
atb
Christoph
Yes. It turned out that it was the trypsin that was too weak. I used 0.25% trypsin and voila, cells detached nicely. I got a lot more single cells and higher cell count. Trypan exclusion didn't show significant cell death either. Though this trypsin is old so it was a little weak still.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence