We are experiencing a problem when digesting the mouse mammary gland. At the end of the procedure (that is the one published by Weinberg group; digestion with collagenase+ialuronidase+dispase/DNAse), we find, a huge number of "objects" (I use objects since I am unaware if they are of biological origin) mostly in the shape of rectangles (like sheets). This stuff cannot be eliminated by pelleting or by filtering at 40um. Since we grow cells in suspension, we cannot even eliminate this contamination by cell adhesion. I know it is difficult, but is there anyone that can suggest the origin of the contamination, or a smart way of getting rid of them? If I can I will attach a photo to make it more clearer.
Hi Matteo
Can you upload your exact protocol and some images of your 'objects' ? It'd really help with the troubleshooting.
We routinely dissociate mouse (and human) mammary glands in our lab and have no issue with these artefacts, however we seldom do suspension culture with the cells (we do colony forming assays and transplants).
I would strongly recommend a read of this article (http://www.ncbi.nlm.nih.gov/pubmed/22644112), as the dissociation protocol you are following is not a standard one used in the field. In short, there are three main dissociating protocols in general use: Matt Smalley's, John Stingl's and Jane Visvader's, and this article compares these. The reason for reading this is that it may be an artefact specific to your dissociation protocol.
Hope this helps.
Al
I often get similar "debris" when I culture primary mammary and prostate epithelial cells., using either collagenase/hyaluronidase or collagenase/dispase (I see no difference in yield between the two enzyme combinations). Normally after a day the epithelial cells will adhere to the plate and these other objects can be washed off. I can usually culture the cells just fine without them becoming contaminated from these materials, but in order to ensure minimum contamination I culture with pen/strep + gentamicin. I also leave the tissues in .02% bleach for 20 min. at room temp before digestion- there is some cell loss but I still get a good amount for cell culture.