Hey everyone,
I've been working with human mesechymal placenta stem cells in our lab. We isolate them directly from a polacenta taken from a C.section. We expand the cells and freeze them at P2-P3. After thawing the vials and growing them for avbout 1-2 passages the medium becomes opaque and the whole flask floor is covered with a net like culture. After confirming that it is a mixed bacterial infection, we removed all the samples and sterilised all the facilities and tools. We received a new placenta and did the same procedure. The contamination reoccured. It is important to note that all the solutions and technics have been used before and they resulted in good yield without any contamination signs, and, offcourse all the work is conducted under sterile conditions.
Do you have any ideas where can this contamination come from and how to deal with it?
NOTE: The red one is PI stained
Have you used new aliquots of FBS/Trypsin/Glutamine/Medium,...?
You could make sterility controls of all your solutions by placing a small aliquot in the incubator.
May also be a contamination source in the filter system of Laminar Flow Hood or in air condition system of the lab. Does the same contamination also occur in other cultures?
Do you store your samples in the vapor phase of your N2 (liquid phase is also a contamination source)?
We performed the solution and media check by placing small flasks in the same incubatore all came out clear.
After the contamination first appeared we changed all the filters including the one in the incubator and in the laminar flow hood.
This is the only kind of culture we are working with and it has gotten contaminated...
We store our samples in liquid N2 bank. We also checked the freezing solution for contamination in the same way as the solutions and they came out clear as well...
Because your contamination just appears after freezing/thawing of you samples, I still assume it's the liquid nitrogen itself (compare Fountain et al 1997 "Liquid nitrogen freezers: a potential source of microbial contamination of hematopoietic stem cell components"). Or it comes from the experimenter.
Maybe you can give a sample of your contaminated cultures to an diagnostic lab, so they can determine which bacteria are in your culture and make an antibiogram. So on the one hand you will see, which antibiotics you can use to treat your samples and on the other hand you'll maybe get a hint, where the contamination comes from.
We did give contamination samples for diagnosis. The couldnt identify the exact strand just said it was mixed bacterial infection. They also found that the bacteria are sensitive to antibiotics that harm our culture...
As for the liquid nitrogen, we share our tank with other labs and we asked them about it and they said they had no signs of contamination.
About the experimenter... we considered this and made a follow up of who was working with the tissue culture,we split and worked on different samples but the contamination appeared in all . We found no corelation.
OK, seems you did really good work in excluding a lot of possible contamination sources.
A very obvious question: Does your sterilization (e.g. pipette tips) work properly?
Do you still have cryopreserved aliquots of the cultures which contaminated later? So maybe you can give a frozen aliquot to another lab, let them thaw and culture the cells and see if the contamination occurs. Or you can give the frozen sample to the diagnostic lab? Maybe you can find out if the contamination comes from the cryoconservation process or from your post-thaw culture.
We changed the Filter in the Gun pippettor immediately after the contamination occured...
Other labs refused to incubate our samples after the contamination outbreak (cant blame them I would react the same way).
As for the micro pippettors, they are inside of the hood and never taken out so we didnt consider them as a possible source. But that might be one thread to follow. I will immediately send them for sterilisation (even though they are rarely used in tissue culture maintenance only for splitting/ trypsinisation)
Thank you for your help!
Have you tried pulling up a second cell line from your liquid nitrogen stocks? If you have already tested the media and re-sterilised your hood etc then that would be the next step, to see if other frozen stocks have an issue or whether it is cell line dependent.
How do you isolate the cells from the C-section sample, and how long are they "on the bench for"? Could it be that the time between isolation and transfer to sterile conditions is an issue? I know that when I had been isolating cells from samples used in non-sterile conditions they were highly prone to infection! If this is the case and it is specific to this cell line, one option could be to run an antibiotic wash on your cells at the end of your isolation procedure, prior to moving into culture/freezing medium.
Good luck!
We are situated in the hospital next to the relevant department. the C section is performed in sterile conditions, the trasportation is in a sterile organ trasportation container, and the isolation is in extreme sterile conditions and includes several steps of 10% antibiotics washing.
we thawd several samples. this problem has been for a while in our labe and we checked all the option we didnt find a correlation to freezing or thawing procedure/ facilities.
But thank you for your advice.
Did you change HEPA-filter of the hood too? You mentioned all the filter were changed. Might be the hood is disfunctional, it is the last common source. Do you have one hood?
We have two laminar hood and the hepa filter was checked and changed recently in both.
Well, than mightbe you should try antibiotic... I will check the hoods leaving an open petri dish from 10 to 30 minutes (with some medium in it just opened before, so guaranteed to sterile). If it is show some contamination, your BSC is non-functional for some reason. You can use LB-agar plate rather (with no antibiotic of course).
Have you screened the placental tissue itself (post-excision and pre-freezing) for bacterial background? Unlikely as it initially seems, on brief overview, there's a reasonable chance that you're co-culturing bacterial flora endogenous to the post-excisional environment (acquired either peri-surgically or from materials used in handling and transport, prior to your tissue processing in hood)?
I have had similar issues with monkey nervous tissues received directly from necropsy - I now take the tissues intact and soak them over night in my normal medium enriched with 10% antibiotic -antimycotic in incubator. The next morning I remove and proceed with my normal isolation and culture using standard medium conditions (1% antibiotic (pen/strep)). You could try adding 1% gentamicin in with the 1% pen/strep.
Larry, We havent performed any screenings. mainly because the contamination started to appear only a year ago. As for previous cells isolation from the placenta no contaminations occured this is why we dint find it necessary, since nothing changed in the V. Section and transportation methodes. If it is necessary to perform a screening assay I would love to know where can I find more relevant information about it (we cant run PCR because the microbiology department wasnt able to determine the strand of the bacteria that is responsible for the contamination , (they called it mixed bacterial infection unidentified)
Julia, Unfortunately in our protocol we must procede with the isilation within 4 hours after the V.section. we do perform 10% antibiotic washing with an incubation time of 10 minuts between different steps. As for the gentamicin the bacteria was found resistant to this antibiotics with 1% and a higher % is toxic for our cell culture.
Hello Jenny
I had same issue before, but I work with rats not human samples. Actually, primary culture and specially stem cells expose to contamination. You should try these steps: change the antibiotic vendor, decontaminate the incubator, make sure you are using the same tips that you used before, make sure the placenta sample does not have any infections such as blood because some times if the sample has RBCs and WBCs, the culture will expose to bacteria and fungus. however, if you see these things in your sample, you need to treat your sample with RBCs lysis buffer before you culture the cells.
Probably the tissue is microbiologically contaminted. A brief washing with antibiotics as you describe definitively is not sufficient to eliminate the microbial load. You should enriche your primary culture with antibiotics. Pen/Strep usually is a good choice, however does not always work since from time to time you have to deal with resistent microorganisms. We worked with skin biopsies and from these experimants we found out that Pen/Strep was ok in >95% of the cases, and it may take several days of antibiotic treatment to get the culture free from microorganisms.
dear jenny,
Placental tissues may be contaminated with naturally existing bacteria, I came a cross some researchers using 1% sodium hypochlorite wash for 20 seconds for the tissues and they all claimed clean cultures with no loss of tissues specially with infected tissue cultures
could be also from the incubator itself or the water bath or the instruments
I too recommend Sodium Hypochlorite.. I have done placental culture in past for epithelial cells.... we could not culture beyond P3 though... but no contamination..
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