I've worked with several types of primary cultures from mouse healty and tumoral tissue. Usually that kind of jelly is constituded of DNA released during the mechanical part of the dissociation process. Unfortunately it works as a "crowding agent" for suspended cells preventing their adhesion on the flask.

Avoid its formation during tissue dissociation try to add DNAse to the trypsin, this really worked for me in the past.

To approach its removal in the already plated cell preparation, the only thing you can do is recover the jelly, incubate it with trypsin and DNAse then block this "digestion solution" using 20% FBS added medium; spin the prep down and plate. I can't guaranty the success but is the only thing I'd try in this scenario.

Good luck.

HI Dan,

that is somsehting strange...anyway, whatever it is it cannot be good ;-) so, if your cell of interest are already attached to the plate then I'd recommend to get rid of the media, do some PBS or media washes until you get rid of that floating jelly you've got.

Good luck!

Hi Luciano,

Thanks for your seggestions. The problem is: the jelly has just packed all cells into it. There is nothing left on the culture flask except a couple of fibroblasts.

Dan

Hi Dan,

Have you tried with DNAse? Sometimes when cells die DNA is released and creates a jelly-like structure that trap cells in it. It happens to me quite often with dendritic cells. Probably it is not your case, but give it a try.

mmmm...that's tricky. Look, it may be nothing. I certainly never saw this particular jelly-like structure before, but have seen funny looking stuff when trying to get primary cultures from tumour tissues....some looked like just fat, other were just fibers (probably extracellular matrix, mucus-mucinous material secreted after plating), but I never worried. So, I if the cells are tangled in those things just keep an eye on it...avoid contamination with bugs and that should do for the moment. Once cells become healthy enough to attach and proliferate, and fibroblast end up senescing you will have a better idea how your precious cells are doing. Give it some time because there is nothing you can do for now. I would not trypsinise them for now as your cells are still getting used to culture conditions without stroma.

Hang in there...they will be fine ;-)

Good luck!

I work on HEK293T cell lines, and some time had some structures exactly like this in my flask, which in the last was found to be contamination and got rid of it with the a new batch of cells, medium and fbs. So keep a check on mycoplasm contaminants and your fbs quality. Secondly take care also while trypsinization many time over trypsinization can cause cell clumps which while replating stays in clump and can show up like these ghost structures.

Good Luck :)

Maybe it is lysed cells

I've worked with several types of primary cultures from mouse healty and tumoral tissue. Usually that kind of jelly is constituded of DNA released during the mechanical part of the dissociation process. Unfortunately it works as a "crowding agent" for suspended cells preventing their adhesion on the flask.

Avoid its formation during tissue dissociation try to add DNAse to the trypsin, this really worked for me in the past.

To approach its removal in the already plated cell preparation, the only thing you can do is recover the jelly, incubate it with trypsin and DNAse then block this "digestion solution" using 20% FBS added medium; spin the prep down and plate. I can't guaranty the success but is the only thing I'd try in this scenario.

Good luck.

If it is mucin you can disaggreggate it using 0.05% DTT or similar concentration of acetyl cysteine in PBS (sterilized by filtration). Mucin secretion is common in the colonic cells I work with. Remove the medium and add the DTT solution to the clumps for 10 minutes then pipette vigorously then centrifuge at 1000rpm for 5 minutes and resuspend in your growth medium.

good luck.

Hi, I guess you may experiencing a type of contamination sourcing from your patient, so I suggest if you have approach to the source of cells, first treat the present cells with high concentration of antibiotics for few days, if you get rid of them, in which you might get your cells also lost. Then you get the cells from the source and work with higher concentration of antibiotic than you are now using. To me the jelly shape, particularly the lower one, looks somehow live material!!!!!

And of course their movement in the culture should be due to their physical structure which attract them together, as if they are from a broken structure gathering together and growing and polymerizing.

I feel think that its a tissue aggregate or cells which did not get separated and formed a clump as tumor cells usually do. I suggest you may try a cockatil of enzymes like trpyisn, pancreatin, collagenase to digest your tissue completely. Also serial digestions work and at the end you can pool in the suspension from all digestions. You should however allow your supsension to stand for a while after every digestion so that the undigested tissue settles and only digested cells are in the suspension which you can aspirate into a new tube containing media. Finally, to get rid of such aggregates if still left, you may use a cell-strainer.. This would definitely remove such clumps.. I have not tried it on tumors but digesting whole hearts. I had clumps when I used only collagenase and single digestion but they disappered on using enzyme cockatil and serial digestion. This also increased my yield of cells.

Hi Dan

I agree with both Luciano Castiello and Sebastiano Curreli, my guess is that it's just DNA from burst cells clumping together your remaining live and dead epithelial/stromal/haematopoietic cells.

We routinely receive and dissociate primary breast, ovarian and prostate tissue (both normal and tumour) and either flow sort these, or culture them, or use them for in vivo assays. Our protocol involves a DNAse step (after collagenase/hyaluronidase and trypsinisation) that we combine with a Dispase step. This sorts out most of our 'alien' gloopy clumps.

Hopefully this helps

Al

I agree with Alasdair Luciano and Sebastiano, Routinely when we dissocate tumor or normal tissu we use DNase to eiminate jelly structure to have free cells;

I think you are looking at the interaction of cancer cells and neutrophils. Neutrophils release actual nets to catch cells, these nets are made out of DNA and contain lytic enzymes along the length; neutrophil extracellular traps.

http://www.pnas.org/content/early/2012/07/18/1200419109.abstract

It is quite easy to see if you have NET's as they have myeloperoxidase. Hydrogen peroxide and ADHP (10-acetyl-3,7-dihydroxyphenoxazine) produces resorufin. Resorufin fluoresceses Ex 530-540 nm and Em 585-595 nm.

If you grow your cells on a plate surface and then add a small amount of your own plasma, you will see your own neutrophils make their own NETs.

I agree with Sebastiano. This is often DNA from lysed cells and can be removed with DNAse. However, it does imply that there are a lot of dead cells that you might want to get rid of on Ficoll/Hypaque or similar density medium, e.g. Lymphoprep.

It could, however, also be a fibrin clot as human ascites is often bloody. In this case you should collect into heparin or citrate and wash the cells by centrifugation. Incorporating a Ficoll/Hypaque density separation into this procedure will also remove the rbcs.

Do you use glasswear petries for culturing or plastic dishes? which one?