I have had consistent contamination in my primary culture of mouse dopaminergic neurons and I am not able to culture the cells beyond 5 days. From visual inspection, I believe its a fungal contamination. I use pen/strep in my feeding media but at this point I am sure its not working. I am thinking of using Gentamicin or flucazone next time. Any suggestions will be great.
The main trouble here is I am not able to find out where is the contamination coming from. It is certainly not the incubator since there are other culture dishes that don't have any kind of contamination. I doubt its the media or its components because our lab follows strict sterile protocols for tissue culture. However, I won't rule it out and next time I will make every component of the media fresh. Either, the contamination is coming from one of the steps in the tissue extraction process or it might be from the litter itself (or the Lab animal facility). I hope its is not from the litter as I cannot do anything in that case. As far as I know my aseptic techniques are as stringent as other labs and peers that are culturing embryonic hippocampal neurons. I am guessing the embryonic culture by its nature are more protected from contamination than tissues from postnatal animals.
I have failed three times and its disappointing. If anybody have experience with culturing mesencephalic mouse dopaminergic neuron and have had similar experience and have resolved it then please let me know. I will highly appreciate your suggestions.
Thanks
Fungizone and
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Hi Maibam,
How old of the pups you used? I met a guy previously who is working on interneurons. He said that he could not get good interneuron survival when using E18 pups. But somehow it is dramatically improved when using E14.5~E15.
Maybe you can try younger pups?
Best,
Yu
In our lab we also set up primary cell culture of ex vivo blood cells. We found that Primocin is great for primary cells and at 1:500 dilution is fantastic for removing all contamination. It is broad spectrum so no need to add pen-strep either.
If I am really paranoid, I even make up my media, add the primocin, then in a class II cell culture hood, filter sterilise the media containing antibiotics through a 0.22 micron filter to really make sure my media is good. After three passages, the culture should be clear.
Good luck!
http://www.invivogen.com/primocin
Thanks for all the answers. I really appreciate it. the pups I use are between postnatal day 2 and 3. I think postnatal cells are more suited for my experiments. I understand the likelihood of survival with embryonic cultures and I have also cultured them before with little to no issues of survival. I am trying to reproduce culture survival of these cells similar to the papers where the labs have successfully cultured dopaminergic neurons from postnatal animals for more than 2 weeks. Also it seems cultures from embryonic tissues are not very confidently accepted in the field of parkinson's research. Therefore, I am trying to get it work with postnatal animals.
Primocin is something new to me. I recently bought Gentamicin and the next culture will tell how well they work. thanks for the information. I will keep posted.
Thanks!
I can vouch for Primocin, good stuff, never had a contamination with it. But then again, neither have I had any with Pen/strep. Are you spraying the pups in 70% ethanol before starting the dissections? The no 1 contamination source is of course from the fur of the animal. Autoclave all instruments every time, and use separate sets for skin and brain. You may already be doing all of this, but thought I'd mention it anyway. Post-natal is indeed more sensitive, but we set up D7 hippocampal cultures on a weekly basis and have never (touch wood) had any contamination of any kind, so it shouldn't be a big problem really.
Thanks for the suggestion Neil. Yes, I spray the animal with 70% alcohol before dissection. I think the culture seems to have fungal contamination which I think is from the animal itself. So I need to take more precautionary measures and be confident from my part at least. I am doing all I can to make the process contamination free and the only one more thing I can do is to add antibiotic in the dissection medium also. I sterile filter my dissection medium and keep it in a sterile glass bottle and most protocols that I have followed just sterile filter the dissection medium but don't add any antibiotics. I guess adding primocin or gentamicin can probably help. I will keep posted. Thanks again.
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