I have been doing long-term cultures of primary cortical neurons for several years, from 2018-2021, with no problem. I use Neurobasal medium with 2% B27, 1% glutamax, 1% penstrep. I would grow my neurons up to 31 days in culture at 30,000 neurons per well in a 12-well plate on a PDL-coated glass coverslip. I would change the entire volume of media on the 4th day in vitro (DIV), and then 50% of the volume every 3 days until the 16th day, after which I would change 50% of the media every 6 days.
Suddenly, at the end of 2021, my neurons died after DIV16. The neurons were beautiful at DIV16, and suddenly died the day after. I thought it was the B27 and glutamax and penstrep that were close to expiring, so I ordered new B27 and glutamax and penstrep and cultured again, but then the cultures started to die after DIV6.
In one of my recent cultures, one coverslip of neurons looked beautiful and just like how my neurons used to look in the past. However, the rest of the neurons in the other wells were dead. I used the exact same dissociation media, same neurobasal + supplements media, and same PDL coating on the same acid-washed coverslips from the same jar on every single well. So, I thought perhaps there was variation in the coverslips, so I washed them more intensively for my most recent culture, but the cells still died on DIV7.
What could be happening? I would appreciate any help.
João Victor Cabral-Costa Thank you very much for your reply! Our building has been without distilled water for a while so I hadn't been able to refresh the supply of autoclaved water/PBS in our tissue culture hood, which may be contaminated by this point. I make my media fresh in small batches and syringe filter it so I think changing the water and PBS in the hood will be my next step, since now our distilled water supply is back. I will hopefully update with good news after my next culture. Thanks again!
João Victor Cabral-Costa Hello again, just a quick update. I had been using half of the recommended media volume in the past just to save on resources (for example, 500uL per well in a 12-well plate instead of 1mL), but after going back to using the full recommended volume of media, I was able to grow the cells up to DIV16. However, they still died at day 17. I believe my incubator is at fault, for we have not kept up on its maintenance since at least 5 years ago. Even the HEK cells I am growing are growing more slowly than usual. I also have to put in a lot more water than usual to keep the humidity up to par. I will try to get it repaired soon and will update this question again!
Edit: Also, I did test the cultures for mycoplasma and they were negative!
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