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Cell line contamination? - (Mar/25/2014 )

I've been working with L6 myoblasts for about two months now. A while back I saw a few strange looking cells mixed in with them and though "eh, I guess the lab that gave us the cells cross contaminated them with Vero." Not a big deal really, as long as they work as host cells that's not a problem. The thing is, I also work with Vero and these things aren't looking like Vero at all really and they're also becoming more numeous. Also, whatever they are, they can't be infected with the parasite that I work with.


Could it be possible that these myoblasts are starting to differentiate? I always got the impression they formed long tube-like structures when they differentiated, not like round striated blobs. Could it be a syncytium? L6 myoblasts are prone to forming syncytium.


Has anyone seen these cells? I took a few pictures with a camera up to the eye piece (our camera scope has seen better days):


Edit: magnification is 200x, the elongated/triangular cells near the top and right are regular L6 myoblasts.

-sir octopus-

These look like they are probably senescent cells.  I'm not familiar with L6 cells, so I don't know if they are transformed/immortalized in any fashion - but in any population of cells that has been maintained for a while, you will get cells appearing that are larger, flatter and usually more vacuolated than the normal cells - these are the senescent (G0 phase) cells and are permanently stuck in the G0 phase.  Lines that aren't immortalized or transformed will have these appear with increasing frequency as the cells approach the Hayflick limit.


I did a little research and it turns out that L6 myoblasts have been immortalized. I spoke with some PhD students in a neighboring lab that are much more familiar with tissue/cell culture than I am and they suggested that they might be dead/dying cells. Could make sense, I seem to see a lot more of these after I trypsinize them, perhaps I'm overdoing the incubation time with the trypsin. But even in a 70-80% confluent culture it still seems like I'm seeing more of these weird cells.


Is there any possiblity that myoplasma or some other bacterial/fungal/viral contamination could be making cells end up senescent? Other people do work with herpes virus in the same hood that I use.

-sir octopus-

Infection can change the way cells behave and look.  Herpes is a plaquing virus IIRC - it would kill the cells rather than change the shape.  Mycoplasma does all sorts of weird things to cells, potentially this could be the problem.  Dead/dying cells usually die within a few hours, so you should be able to see them blebbing etc as they apoptose.


The thing about these weird cells is the culture has been going for two days since the last subculture and they still look pretty much the exact same, no noticible blebbling going on. I tried looking at them under 400x to check for some tiny contaminants, but some idiot never tuned off the light for the scope and burned out the bulb I just replaced 3 weeks ago, so no luck on that. Their numbers haven't really increased at all, just stayed the same. The same L6 cells that were pictured are growing just fine, with no more of the weird cells showing up as far as I can tell. Could over trypsinizing the cells possibly cause this?

-sir octopus-

These cells usually disappear after few passages. I've had them before.