Dear Anastasya Birger

In my opinion, it is not the best case scenario to have both hiPSC and animal primary cell culture in the same room esp if you are maintaining hiPSCs in antibiotic free conditions.

if possible keep the hiPSC culture separate from primary cell culture.

You are already following several steps to avoid contamination.

Here are a few more points that are crucial to maintain sterile conditions based on my experience which you may choose to add to your protocol (if you are not doing so already):

1. switch on UV everyday before and after use of the cell culture bench for 1 hour

2. clean the bench, water bath twice a month.

3. wipe the surface of the microscope and the workbench around it every time you wish to check your hiPSCs

4. after switching on the bench, wait till the airflow stabilises before you start working.

5. make sure the bench inside is not overcrowded with tip boxes etc so that the airflow works properly and nothing is blocking the air flow. Also, a good idea to check if the filters are too dirty or need replacement.

6. make sure that while changing medium - all cell culture plates and medium are placed properly inside the laminar air flow and not kept towards the edge of the bench. Also, avoid crossing over your hand over the cell culture plate while it is open.

7. make sure that only sterile pipette tips and aspirators are being used inside the bench

8. wear proper gloves and if needed change gloves frequently.

Perhaps these YouTube videos might be helpful to get some more useful ideas-

https://www.youtube.com/watch?v=ZnUW1N-JJz8

https://www.youtube.com/watch?v=q_C6xq7j-kg

good luck !

Dear Anastasya,

Now, our lab also often generates MEFs from E13.5 mouse fetuses.

Our clean bench for human iPS cell culture is also used for mouse dissection and MEF culture. However, no noteworthy contamination incidents have occurred. We have confirmed that human iPS cells are clean for mycoplasma as well. (Of course, as Shambhabi mentioned, I think sharing should be avoided.

We add the recommended amount of Gibco's penicillin-streptomycin reagent to the culture medium.

I don't think the environment or equipment is the cause of your trouble. Are your reagents or cell stocks contaminated to begin with?

Best,

Thank you very much Shambhabi Chatterjee! We do all these actions, but I really appreciate that you stressed their importance.

Narumi Uno, I greatly appreciate that you shared your experience! I am testing right now every reagent I use. I have seeded samples on the dish plates with LB without antibiotics. For now, I can't find the source. But cells are dying and I clearly see moving particles in the culture, and media turns yellow very quickly.