How can I get rid of podocyte cell culture contamination?

Hello there.

 

I've been having a really hard time with my cultures of human immortalized podocytes. Everytime I put them under differentiation conditions, they die because of contamination.

 

For more context, this culture is expanded in DMEM F12 10% FBS at 33°C until confluence. Then, for differentiation, it's subcultered into some plastic previously coated with laminin/fibronectin and maintained with RPMI 1% FBS 1X ITS at 37°C, with media changes every other day.

 

When the cultures are expanding they're fine, but when I start the differentiation they die before a week, that's to say, about the second media change. Cells look detached, media looks cloudy and slightly basic, and I've seen small dark dots, so I'm guessing it's bacterial contamination.

 

No other culture at 37°C gets contaminated, we've prepared new media, new PBS, I've thawed several vials frozen at different times and everytime I get the same results.

 

So, do you think it's bacterial contamination? Is it possible that the source of contamination is the laminin/fibronectin solution or the ITS? Obviously the problem starts when that is used (I have some other podocyte cultures that are not contaminated when expanding, even for weeks) and those are the only reagents that haven't been changed, could bacteria resist there?

 

Than you in advance