We are growing amniotic mesenchymal stromal cells in culture. The cells are isolated from a term placenta, delivered via C-section, using collagenase. We culture the cells in 10 % FBS DMEM (0,1 % Gentamicin). The serum we use is not filtered. The isolation is carried out under strict aseptic technique.
I have attached a photo of the culture 16 days after isolation (passage 2). Numerous black dots can be seen (some I marked with red arrows).
We also carried out a DAPI stain (see attached), but it was negative for Mycoplasma contamination.
Are the black dots from contamination with bacteria or fungi? Or are they simply cell debri? The cells do not grow very well, we are worried the black dots could be connected to the stunted cell growth. Also, the cells adapt a wide shape, instead of the slim fibroblastic morphology they should normally maintain.
Today we also placed samples of the serum and the medium we are using into an incubator (aerobic and anaerobic conditions), to see if the black dots will appear or not. I will post updates 5-7 days later.
Any ideas will be appreciated. Thank you!
Bacterial contamination appears to be the source of the problem. Before using your media, I recommend filtering it using a 0.22um filter. In addition, an antibody cocktail may be able to save your culture. Please read the article that goes with it. Good luck!
Hello Paulius Valiukevicius
You may also refer to the article below.
https://www.biorxiv.org/content/10.1101/366906v1.full
Best,
These are black particles if not vibrating then cell debris otherwise mostly due to bacterial contamination. Filter the media and FBS, add Penstrep. It will go off.
Don't assume the black dots are microbial contamination (although placenta is hardly 'sterile'). Recall that culture media is essentially salt water; and that in water (aqueous) salt dissociates into charged ions. What do ions like to do? They associate with counter-ions! Are there counter-ions in this environment? Yes! Both your cells and serum supplement include vast concentrations of charged counter-ions.
I have always considered 'black dots' (which you'll note are heterogenous) are simply multi-molecular aggregates (i.e. polysaccharides, proteins, lipids) that assemble naturally in situations where the entropy of aggregation is lower than the entropy of remaining in solution / aqueous phase. Consider that the ions are the 'glue' that holds these aggregates together. Changing the physicochemnical conditions (pH, T°C, [Conc], macromolecular crowders, etc), of the media/culture will also change the characteristics of the 'black dots'. Have you noticed how they come back a few days after sub-culturing, coincident with the media changing colour and cells growing/dying?
I find it difficult believe that your 'black dots' are the reason you cells do not grow well. Being primary isolates, they now find themselves in a totally alien environment, bathed in serum; serum (liquid component of clotted blood) is the bodies universal alarm signal for injury! Your cells are in a state of shock and awe! Would you be happy find yourself in a similar, unasked for, situation? How might you respond? Flight, fight, or acquiesce?
- Badges
- Science topic