I am doing whole cell patch clamp electrophysiology on acute cultures of midbrain dopaminergic cells. I normally start doing my experiments on DIV 7 and go as far as DIV 12. However, I have observed that the cells are not happy in my bath solution (Ringers, 298 mOsm) after DIV 9 and they tend to swell and die within 20 -30 mins of sitting in the bath solution on the rig. This is a problem that I have examined closely in my cultures and I have measured the osmolarity on each day after DIV 7 for many batches. The osmolarity increases as the culture grows old. This becomes a problem for patching and as a result it leads to a bad day on the rig. I have even started using ringers solution with different osmolarities for each day in order to get as close as possible to the what it was for the media.
I think, and may be it is obvious, that these dopaminergic cells are releasing dopamine and other macromolecules in the extracellular medium which contributes to increase in osmolarity. However, I am surprised to see a change in osmolarity from around 290 mOsm on DIV 7 to 290 mOsm on DIV 10. I wonder if this is normal or something is not right in my culture. If anyone have had similar experience and have any insight on it then please let me know. This issue has made patching really difficult.
Thanks
Hello Maibam:. Are you sure that it's all related to changes in osmolality of the media but not to general deterioration of neurons in the culture over time?
Hi Victor,
Thanks for your response. I have not ruled out generation deterioration of cells overtime in culture but as I have observed significant changes in osmolarity after DIV7 makes me suspect if it is the problem. Though it is hard to quantify changes in cells health overtime just by looking but I can say that the cells in cultures look fine even after DIV16. This is coming just from crude observation though. My justification here is swelling of cells which I think is due to my bath solution having less osmolarity then the media where the cells were before the experiment. Let me know if you buy this justification. Thank you again.
Maibam: if cells are healthy than they should accommodate to the bath solution osmolarity within 20-30 min (max) after transfer from the culturing media. However, based on my experience, many neuronal preps are VERY sensitive to the DIFFERENCE in osmolarity between external (bath) and internal (pipette) solutions. In my hands, the best survival in acute patch-clamp recording was achieved when internal solution had osmolarity 5-7 mOsm lower as compared to the bath
Hi victor,
I am patching my cells this weekend with a new internal solution. I will share what I get and observe with the cells health.Thanks again for your suggestions.
Hello Maibam, I am also curious whether you feed your cultures every other day or so?
I feed them only after DIV 14 when I am doing experiments that far in the culture. I have seen some labs feed them 2-3 times a week but I wonder if its is necessary to do it that frequently for the dopaminergic neurons. You think I should feed them more often?
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