Dear All,
I work with human MSC primary culture. I and my colleagues have observed suspicious contamination in our culture (different MSC sources, two different incubators). Can you help to identify the kind of contamination? What should we do?
That concerns us a lot - we observed that cells did not want to attach to surface after passaging. we noticed worsen morphology of our cells, changes in housekeeping genes expression in PCR.
What have we done so far?
- colometry assay for Mycoplasma - no detection
- PCR - no detection,
- microbiologic tests for molds and yeast - no detection,
- always filter 0,2 um medium with plate lysate before use,
- used antibiotics: firstly mix - penicillin-streptomycin-amphotericin, secondly - double the concentration of that mix, then - pure amphotericin; no results,
- before work, sterilize hood with UV; every day at the end sterilize room with UV; using alcohol and anti-mycoplasma cleaning tissues and liquid detergent,
Can someone help us?
Photos: primary culture of WJ-MSC in magnification x10 and x20. Black dots may be bacteria (they were slowly moving).
Hello
If you doubt there is bacterial contamination please confirm that too by streaking LB agar plate with a few micro litres of culture medium containing cells and incubate the agar plate in the incubator at 37 degree celsius overnight. Check for bacterial colonies the next day.
Regards.
Dear Agnieszka!
If you have done all these procedures and did not find contamination, according to the photographs, these may still be dead cells due to the type of apoptosis, when small apoptotic talci are formed. Based on your description, cells do not grow, do not attach, but die. Apotosis is a physiological, programmed type of death, and therefore your cells do not like any factors. Check media, serum, supplements to ensure all reagents are fresh. check the culture dishes (dishes, plates, vials), you may have used other manufacturer's dishes with different adhesive properties. Primary cell cultures like to grow on highly adhesive plastic.
One more thing: Your cells can just get senescence. In this case, their adhesion also decreases, the expression of surface markers changes, and increased death is observed. In this case, it is better to replace the culture, to defrost new cells.
Malcolm Nobre Thank you for reply! We did agar assays - we did not have any colonies after incubations
Alexandr Chernov Thank you for your suggestion! Those cells are freshly isolated from tissue - passage 0. Usually we do not observ senescent cells at this stage of culture (donor age is briefly after birth as umbilical cord is the source of MSC).
About apoptosis - if it is true, it would concern us a lot. We try to find the reason for the apoptosis. We use highly adhesive plastic, so far we dont have any complaiments about it. However, we see similar situation (lack of adherence) on plastic surface on different culture dishes (6 well plates, flasks) - from different manufacturers.
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