3 replies to this topic

[elpollodiablo]

Hello,

I've recently bought a batch of progenitor cells isolated from a human fetus from DVbiologics. When culturing them however, I've run in to some problems. There seems to be some kind of contamination inside the cells (see attached pictures). Small black dots that seem to prefer to reside around the nucleus. In some cells there are many, in some few and in some none. I think they are visible in the media outside the cells as well, but that could just be cell debris. There is no change in pH and the cells are quite healthy(~95% viability).
I've taken up a fresh batch from the freezer: they appear. I've sterile filtered everything, yet still they appear. They seem to be impervious to pen-strep. They do not stain with DNA staining dyes and the extracellular ones (debris) seem to ignore the concept of alkalic lysis. When a sample of cell culture media is incubate in sterile LB media, nothing has grown after a few days.
When appearing extracellularly, they appear to vibrate, but inside the cells they seem to be stationary.  They are driving me mad! Does anyone have any thought what this might be??

 speck1.jpg   747.01K   94 downloads
 speck2.jpg   640.93K   108 downloads
 speck3.jpg   660.58K   70 downloads

Edited by elpollodiablo, 23 September 2010 - 05:52 AM.

[leelee]

I couldn't really see them very well in your pictures, but I don't think it is contamination.

You don't see anything in LB broth, and you haven't seen an increasing number of them in your culture, nor a pH change.

Keep in mind that just because something is vibrating in your media, doesn't mean it is living. Brownian motion will cause non living debris and other small partices to vibrate in media.

[Rsm]

Looks to me like inside the cell. Could be melanin granules... Are you sure you didn't buy melanocytes?
I wouldn't worry too much either.

cheers,

rsm

[elpollodiablo]

Thank you for your input. The black things are certainly inside the cell. I fear some sort of intracellular organism, but I can't find anything about that in the litterature (at least not as a common cause of cell culture infection). I'm pretty sure my cells are not melanocytes (if they are, the company has screwed me over good), but perhaps some sort of other granule could look the same? I've only worked with HEK cells so far, so I don't really know what other cells types should look like. This post (http://www.protocol-...posts/4305.html) also made me very uneasy!