How can I get rid of podocyte cell culture contamination?

[Andres]

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

[OceanBreeze]

You don't mention any testing that you have done so there is no way to make any sort of determination about the contamination.

Since you are say the problem starts when Fibronectin coating is used, that is probably where you should start your investigation.

I recommend you visit this site and make sure you are following the protocol correctly and all steps are performed in sterile conditions, obviously.

Other than that, you will need to do the work to troubleshoot and test everything as recommended by this site.

Good luck!

[Vmedvil]

On 9/19/2023 at 11:34 PM, Andres said:

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

 

8 hours ago, OceanBreeze said:

You don't mention any testing that you have done so there is no way to make any sort of determination about the contamination.

Since you are say the problem starts when Fibronectin coating is used, that is probably where you should start your investigation.

I recommend you visit this site and make sure you are following the protocol correctly and all steps are performed in sterile conditions, obviously.

Other than that, you will need to do the work to troubleshoot and test everything as recommended by this site.

Good luck!

When I was reading this, I thought pretty much the same thing as OceanBreeze, the contamination cannot be determined by the description that Andres gave, and everything needs to be checked. I was thinking maybe it is being infected by the either the bacteria or plasmid the fibronectin coating was produced in, and the fibronectin not being properly purified but there is not enough information to confirm or deny that hypothesis, so I Dunno either. Another thing that was bouncing around in my head was possibly a fungal infection considering the "dots" are black, but I am not certain about that too. Overall, Hell if I know...