Hello everyone,
I'm a lab technician and I have a recurrent problem in cell culture : since a few months I have a viscous pellet when passaging the cells. I only have NIH 3T3 Src, a murine embryo fibroblast cell line (adherent), and sometimes -not always-, after the centrifugation step at 900rpm for 4 minutes (these parameters are the same for everyone in the culture room), my cells are agglutinated into a non identified viscous fluid... Which makes them difficult to resuspend into the 2mL of new media. The subcultured cells don't adhere well to the surface of the flask/well and end up dying, floatting to the surface.
Basically i can't do any further experiment because of this problem.
I asked everyone in my lab, and none of them really have an idea what causes this. I happened to one colleague once but she doesn't remember the conditions that caused this viscous pellet.
Here is a list of the solutions/parameters i use for a simple cell passaging:
Media > DMEM 4,5g glucose + 10% FBS + 1% antibiotics (or not, when I'm about to do a transfection that requires no antibiotics)
Physiological washing buffer > PBS 1X
Trypsin 0,05%
Time into the incubator (37°C) : 3mn
Ratio trypsin/media : 2mL trypsin for 4mL media
Do you have any idea about what causes this, and/or have you experienced that before ?
Thanks a lot,
Gaëlle
Dear Gaelle,
One possibility is that the viscous substance is DNA released from lysed cells, possibly from too much time in the trypsin or rough handling of the cells. If you have ever dissociated tissues for primary culture, you might have encountered the same problem if your dissociation enzymes are not inactivated completely. Of course there is more cell damage when you are triturating tissues into single cells, so many of us add extra inhibitors (e.g. soybean trypsin inhibitor) and DNAse to avoid this problem.
I don't use 3T3 cells, but for adherent cells I have detached by using trypsin (rinsed first with Versene rather than PBS) I don't just use a standardized time. I observe the cells instead, and resuspend the cells in fresh media when they have detached. Usually for my astrocytes it takes 10 minutes (T75 flask with about 11 x 10^6 cells; 2.5% trypsin), but I check them at 3 or 4 minutes, then again at 7 or 8 minutes. This is particularly true if I have a new batch of trypsin, since I have found the activity can vary from batch to batch.
For my protocol I add 2 ml of the trypsin to to flask, slightly tip the flask to make sure that all of the cells are coated, but then remove the excess trypsin. I resuspend my cells in 10 ml of media very gently before centrifugation, since they are fragile. While my trypsin is more concentrated than yours, you might want to increase the ratio of media to trypsin so that you are sure that it becomes inactivated and the cells are well cushioned. Or you might try increasing the protein concentration of the media used for centrifugation, either with BSA or just more FBS.
Good Luck!
Jill
Thanks for your answer, so I just passaged my cells this morning and the problem occurred again,even though i slightly changed the conditions as you said (and as some of my colleagues suggested me) : only 1'30 in the incubator with the trypsin instead of 3', it was actually enough to detach my cells, and a lower ratio trypsin/media (1,5mL of trypsin 0.05% + 6mL of media 10% FBS)
The thing is i realised i used an old bottle of media today (no opening date on it), not on purpose of course, and this might explain that the trypsin wasn't inactivated and the excessive lysis action onto the cells. So maybe this time that explains the problem.
I agree with you, the viscous substance must be DNA.
To answer you it's not a primary culture, it's a stable cell line (passaged 10 times before), and no one in the lab is using something else than PBS 1X to rinse nor add extra trypsin inhibitor, yet i'm the only one to have this problem.
Hi Gaëlle, I agree with Jill in that that you have lysed cells and DNA causing the problem.
I have seen this happen when detached cells and cell pellets are pipetted up and down too fast. The shear forces generated by pipetting can be enough to lyse cells. Air bubbles in the pipette containing cells can enhance the shear forces.
If you are the only one in your group having this problem, then observe how other people pipette the cells after trypsin treatment and how they resuspend the pellet.
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