RWPE-1 is a prostate epithelial cell line. I use Keratinocyte-SFM with recombinant human Epidermal Growth Factor (rhEGF) and Bovine Pituitary Extract (BPE). It doesn' t include Pen./Strep. antibiotic. Hence cells are open to be contaminated. What can I do except aseptic conditions (like using sterile serological pipette, petri dishes etc.)? Are there any special antibiotics for them or another approaches? Who has any experience with that cell line?
Hi Mert
RWPEs are no different from any other mammalian cell line when it comes to susceptibility to contamination. It's all a question of technique and, as you say, using sterile equipment such as pipettes and flasks.
Pen and Strep don't offer much protection in truth and it is not difficult to run without them or any other antibiotic - my lab has been antibiotic-free for almost 20 years.
Don't second guess your ability - just DO it!
Good Luck
G
I don't have any experience with the cell line or the media, but is there any reason you can't add antibiotics? Also, you might want to try adding some disinfectant to the water such as Incuwater or an anti-fungal such as fungizone. Couple that with good aseptic technique and you'll probably be ok.
Actually, Grant A has a good point - one key aspect of aseptic technique is to look after your incubator, cleaning it from time to time, using autoclaved water for the humidifier tray (if it has one) and so on.
However, he and I disagree on the topic of antibiotics - sterility in cell culture is a matter of technique and we run antibiotic free (and that includes new undergraduate students and interns).
As to *why*? Well, when I was a lad doing my PhD and first post-doc back in the 80s, there was a fad for moving from a very labile antibiotic such as P&S to more stable ones, mainly Gentamycin. WHile this certainly kept bacteria down, it led to a real issue with what I called "grumbling mycoplasma" - low lever endemic mycoplasma infestation of almost all the cells. Unlike bacteria, mycoplasma are not readily visible, nor will they overgrow a culture (the "medium-becomes-custard" phenomenon). Consequently, cells "go off", results drift, they look "sick" and data vary more than they ought.
It was for this reason that I went antibiotic free and have prohibited their use in my own lab since I have been running one. WHile I can't speak for everyone, my impression is that people rarely think about mycoplasma now and this is a mistake.
Good Luck,
G
ps - hey - nice to meet another Grant, Grant
Hey Grant, nice to meet you too I see you too have spent in Scotland!
I didn't see your first answer, I'm assuming we must have posted almost simultaneously but I agree entirely about antibiotics usage, but what I was questioning is why, if Mert was worried about contamination, couldn't he use them?
As you point out, mycoplasma is something overlooked almost these days and complacency about infection is rife as people assume antibiotic media will take care of most things.
Good aseptic technique and clean incubators and you should be fine!
Grant
Hi Grant, I usually use RWPE-1 cells since 5 years and I always add the Penn/Strep at 1%. Moreover I use also sterile aseptic techniques and try to keep clean incubators and biological hoods:
Good luck: Nico.
Firstly thanks to everyone for your answers. Especially Grant Gallagher, thanks for your informative answer. We are sensitive for cleaning of incubators, laminar flow and using autoclaved water for the humidifier tray in lab. We also check mycoplasma contamination in every 2-3 passage. I just wondered whether there are alternative ways or not by who has more experience than me.
Checking for contamination every 2-3 passages seems quite excessive. I'd worry about technique if the contamination rates justify that kind of screening.
I agree with the Grants above that antibiotics are no substitute for good technique and a clean environment. Our lab also is antibiotic free and it works well (and we are a half-yeast lab)!
However, if you are set on antibiotics I would recommend using pen/strep plus Plasmocin prophylactic to prevent mycoplasma contamination.
Don't worry Robert :) In lab. we use antibiotic because of that contamination occurs very rarely. But as Grant Gallagher said, mycoplasma contamination can be missed, so we check it in every 2-3 passages. It doesn't mean contamination occurs much. (It means we careful so much). Generally, the results are clear.
Thanks for your suggestion Robert. I will also check Plasmocin.
I have to applaud you for that kind of tenacity...that is an incredible amount of screening! But it is a good practice.
Best of luck!
It is clear in manuals "Add antibiotics, if required. We recommend Gentamicin at
5 μg/mL."
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