HUVECs - cells detach after 2 days (Nov/25/2008 )
Hello friends,
I am breaking my head culturing HUVECs since last two weeks. I am not a new to HUVECs. I have been using them since last 2-3 years. But this time I have repeatedly failed on culturing them !!! heres the problem.
# new fresh stock (checked cell viability--more than 95%)
# .2% gelatin coating (chekced DPBS and gelatin for sterility)
# withing a half an hour, cell attach very well
# the next two days, cell grow very well (achieved 70% confluency)
# the third day, cells either start to change its structure or already start detaching ( I am expecting tight confluence, as previous days)
When I passaged cells those in second day, failed to grow (attach).
I have been losing many stocks, with no improvements ---even after checking media, coating, and cells. Please help me why my HUVECs cant grow more than 2 days, more than 60-70% confluency????
Thanks in advance..
Hi Thapa, firstly i must say that i dont have much knowledge of HUVECs but have good experience in general cell culture especially when dealing with sensitive cells like your HUVECS seem to be, as i have been working with primary cells for some time now and have had issues with cell viability after passaging.
Firstly i dont think the problem is due to the limited life span of the cells as you said you have worked with them before for longer without any problems (although i think i read somewhere before that they can be doubled at least 16 times so unless your close to this passage number this should not be the cause of your problem). As the problem seems to occur during passaging steps i would be more inclined to think that the cell death comes during one of these steps.
So for possible soultions, firstly have you recently bought a new vial of trypsin? is it made up to the same concentration as before when the passaging worked? does it contain EDTA as before? have you increased the duration the cells are exposedon your trypsinisation step?
Also have you used any non-trypsin cell disassociation methods?
Such as scraping the cells instead of trypsin (Sigma CLS3010) or maybe enzyme free cell dissociation solution (Sigma C5789)?
Firstly i dont think the problem is due to the limited life span of the cells as you said you have worked with them before for longer without any problems (although i think i read somewhere before that they can be doubled at least 16 times so unless your close to this passage number this should not be the cause of your problem). As the problem seems to occur during passaging steps i would be more inclined to think that the cell death comes during one of these steps.
So for possible soultions, firstly have you recently bought a new vial of trypsin? is it made up to the same concentration as before when the passaging worked? does it contain EDTA as before? have you increased the duration the cells are exposedon your trypsinisation step?
Also have you used any non-trypsin cell disassociation methods?
Such as scraping the cells instead of trypsin (Sigma CLS3010) or maybe enzyme free cell dissociation solution (Sigma C5789)?
Thanks cotchy for your response.
What I thought last week is similar to you. Then this week I took new stock, grown in new flask with newly/freshly prepared gelatin and DPBS (for coating), but the results are still the same.
Day 1: pretty good percentage of cells attached to the coated surface and I changed fresh medium after 3-4 h to remove DMSO, dead cells and/or stock medium.
Day 2: Cells were very healthy and growing slowly. Just checked in the morning and evening.
Day 3: Media changed ....but after few hours when I saw under microscope, cells are already started to detach. ...?????? Ohhhh noh/ what does that sense?????
Again, there is no trypsinization steps since this time I took fresh stock.
I have really no idea, what is going wrong??
Finally, its alright with my HUVECs. Very interestingly changing of Glutamax resulted good growth without detachments. It was just 3-4 month back I aliquated Glutamax and stored them at -20. And it is said that Glutamax is stable form of L-glutamine, but it gave me lots of tenson for 2-3 weeks, heavy loss of time and money. Not imagined before, glutamax can cause such a big problem (the strange phenomenon)....anyway happy with growing HUVECs at last .