I don't know if this is contamination?? - (Jan/18/2010 )

Hey guys,

I got an aliquot of a (commercially unavailable) cell line before xmas and have been attempting to grow them up this last week. Upon recovery from nitrogen hardly any cells stuck and I probably only had <5% viable cells. Of course, I did not freeze them myself and they were shipped internationally so this is not a huge deal, I can live with it but it might be a clue here.

I removed the dead non-attached cells the next morning and refreshed the media thinking that would get them going. Next day, more "dead cells" so i did the same thing. However when I saw the same thing the next day, I started to get interested. The reason is that there was not enough live cells on the plate to generate the amount of "dead cells" that I was removing and the cells certainly do not grow fast enough to replicate and then die...for example. I'm thinking something is growing and it cannot be my cells!

I then removed the media today and spun it down and plated the pellet into a single well of a 24-well plate. Only a handful of the "dead cells" were in the well but my god by the end of the day, they had proliferated like mad and there was thousands of these things in the well. Some of them were even stuck to the bottom of the well.

This immediately alerted me to some form of contamination but I have never seen anything like it before. These things are quite large and just look like dead cells....literally, except their roundness is not as smooth. Inside the little balls, there is grainy blackness. There is no change in media colour or turbidity and no troubles in any of my other cultures. I do not use antibiotics at all.

Is this contamination or just a screwed up aliquot of cells going haywire? Perhaps cross contamination of another foreign cell line? Yeast, mould, even bacteria? But remember no media changes.

Why don't you add pen/strep to your media? It usually protects against contamination.

Also, could you add a picture of your cells in the morning and one at the end of the day?

Hi Dukey,

Just out of my curiosity, is the cell anchorage dependent or suspension cells?

A picture of the cells would be very helpful.

Thanks

stylothecancer on Jan 21 2010, 03:11 PM said:

Hey yeh the cells are very dependent on attachment and are also very dependent on cell to cell contact. I will try and get a picture up of the cells. It is very odd and is only affecting these cells. I received two vials from another lab and as soon as I thawed them I saw that at least 50% of them were what I considered to be dead. They just looked like apoptotic cells. Some begin to attach and then spread and then they just die slowly over the course of the next few days. To me now I am thinking it is a classic freezing down problem. I have contacted the sender of the culture and he has informed me that "on rare occassions they have seen something very similar". Perhaps it is a problem with their freezing down procedure. Some cells are growing but it is very tough for them to get going. I will get some more cells and see what happens. I have not ruled out contamination!

Here is the pics, high and low power:

It looks like apoptotic cells... It's certainly neither yeast nor bacterial contamination.
Sorry that I can't be of further help...

Cheers,
Minna

Hi Dukey,

From the pictures your cells seems like they need more time to attach.

I have this experience before particularly if i received the cells from other lab.

I managed to solved this by:
-upon receiving, thaw the cell, and then centrifuge them to remove all the cyro medium they used.
-then, seeding them into 2 flask if possible; one with the normal FBS% u normally use, one with slightly elavated FBS%
-the next day, please don't remove the medium, but just monitor if there is any contamination.
-if not contamination occur, I would add more growth medium into each flask,
-then, i will just let it grow inside for several day until they attache.
My worst experience is, i need to wait for them to attach for two weeks.
of course, you would found that the cell that are floating is not really healthy as been shown in your picture, but most of the time, I manage to get them attached, provided they are not contaminated upon freezing in the sender's lab.

Good luck.

stylothecancer on Jan 28 2010, 02:38 PM said:

This is good advice, I have been thinking similar things recently. I suspect that I have been disturbing them way too much and perhaps just need to give them time to chill out. I was worried that if they were dying cells, they would affect any living cells so I was washing them away. But perhaps my washing was affecting the living cells. Crazy!

Doesn't look like contamination to me. It looks like the cells may not like the tissue culture plastic. Try them in flasks from a differenr manufacturer, they may adhere better then.

Out of interest what cells/type are they?

Minna on Jan 27 2010, 10:50 PM said:

+1 - apoptotic cells

Re: I probably only had <5% viable cells.

very low.

i think you must elevate FBS% in two times (10%) and add pyruvate (first of all)

think after it about growth factors for your cells. What kind of culture?

bacterial contamination always will decrease Ph (from red color of medium to yellow)

good luck^)

I have seen this also with my cells. Give the cells a day or two more to attach before changing the medium.