Hi, I am culturing cortical neural cultures derived from P0-1 rats. Cells are plated onto poly-D-lysine coated dishes and survive nicely in NeurobasalA + B27 + GlutaMax + PenStrep - 50% of the media is replaced with fresh medium every 3-4 days.

I use the cells between DIV19-21. I am looking at the extracellular vesicles (EVs) they secrete and therefore before adding treatments I have to do a full media change in order to remove the endogenous EVs - I remove exisiting media, quickly but gently add fresh, warmed media containing my treatment (a neuroprotective peptide) and return to the incubator for 24 h. Culture dishes were chosen for treatment because they looked beautiful and happy on the day of treatment, however, when I check them at the end of the 24 h they are clearly unhappy/dead. I have ruled out contamination of the media as the same media is used in younger cultures and does not cause cell death.

The issue seems to be associated with older cultures, e.g. DIV15+, as sometimes even the half media changes cause this issue, but prior to DIV15 the cells are not affected by media changes. Changing media less frequently after ~DIV14 has helped my cells last longer, but the media change required for the treatment itself still causes the problem.

I am wondering if others have encountered this problem. I have read papers (e.g. Driscoll et al., 1991; 1993 in Journal of Neurochemistry) suggesting that glutamine/glutamate is the cause the cell death observed after media changes - and wonder if I should be leaving GlutaMax out of the media at these later DIV. Alternatively, could the PenStrep be a problem at these later DIV? I have read that some only add it in the early days of culturing and then stop including it later on. The Driscoll paper suggested that PenStrep actually exacerbated the problem. Any advice or suggestions would be appreciated!