I have attached images below of what our well plates are looking like for iPSC culturing. Most recently being day 8, where we found potential contamination. The media looked slightly yellow and there was debris floating on top.
We are not seeing the expected colonies form and there is a lot of background in our images. We used Corning Matrigel Matrix, mTeSR™ Plus, and iPSCs. We changed the medium every day and used ROCKi on day 1 and day 2.
I am new to cell culturing and I wanted advice on how I should plan to go about determining what exactly is wrong with the cell culture.
The media yellowing indicates acidification. The debris could be damaged matrigel (can happen if pipetting is too rough). If you suspect contamination consider taking a smear and plating it out on LB agar to look if colonies grow.
Dear Aditi,
The images arent very clear. But looks as though you have bacterial contamination and the cells appear dead.
I would advise you to start fresh, with new matrigel aliquot for coating and revive new hiPSC vial and use freshly prepared media.
All the best.
Thanks.
Prasad.
Dear Aditi,
Please verify for bacterial contamination to rule out any shortcomings in your culturing technique. This can be accomplished visually by observing the contaminated media under a microscope and looking for motile bacterial cells. Alternatively, you can spread a few drops of the suspected contaminated media onto an LB agar plate and incubate it overnight. If bacterial contamination is confirmed, I suggest thoroughly cleaning your biosafety cabinet, pipettes, running a sterilization cycle in your incubator, and strictly adhering to the working protocols of a biosafety cabinet.
If the issue is not bacterial contamination, troubleshooting stem cell maintenance is a different ballgame altogether.
Best,
Hi Aditi Kumari,
like others suggested, you should check for bacterial contamination. Furthermore, I also suggest to regularly check for mycoplasma contamination (e.g. using PCR). We add MycoZap™ Plus-CL (Lonza, Catalog#: VZA-2012) to our iPSC cultures to prevent contamination.
Good luck!
Selene
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