Hi,
We are using mouse primary hippocampal neurons in our lab and they usually work great until 14-16 days in vitro (Neurobasal+ or BrainPhys media, neurons with astrocytes). We usually have more degraded cells after 16-18 days, so we prefer using the cultures for our experiments before that. I was wondering if someone has been working with such cultures 20-30 DIV. Are there perhaps some special techniques that apply to mouse cultures and not those from rat primary neuronal cultures?
Thanks for the info, Attila Szücs
Use a chemically defined medium. Originally described by Romijn et al. Neurosci and Biobehav Rev 8: 301-334. Look also into Marani E (1988) Neuroendocrin 48: 445-452. Large farmaceutical firms have produced these chemically defined medium (I don't know the best nowadays, ask others). We succeeded up to 48 days culture. Note that reduction of the amount of neurons is dependent on surrounding (optake O2, reducing CO2, humidity etc, thus your incubator) and refreshing of the media.
Success and remember it lasted months before we had trained ourselves to keep longlasting cultures.
Dear Dr, Marani,
Thank you for your suggestions. We will try to modify our culture media by adding more vitamins. We have good results with BrainPhys media, the cells are usually very healthy for 14-18 days, but problems start to appear after that. I hope the added chemicals will also help.
Best regards, Attila
Dear Attila,
In our lab we prepare long term primary mouse hippocampal cultures over astrocyte feeder layers following the Banker's protocol (https://www.nature.com/articles/nprot.2006.356) that easily surpass DIV20 and typically reach DIV30 or more. We prepare a N2 neuronal media with Neuropan 2 supplement (https://www.pan-biotech.de/neuropan-2-supplement-100-x.html?___store=en).
Also how the coating the type and method of coating is extremely critical. If the coating is not stable for the intended culture time then the neurons won't reach the intended time either.
Cheers,
Dear Sebastián,
Thank you for your suggestions. We will look into this protocol.
Attila
What age mice do you isolate your cells from?
I culture from P-0 mice in Neurobasal with B27 and glutamax plus 1µM 5-FU, I do a full media replacement at DIV7 and a top-up of approx 120µl on DIV14. The cells are good for recordings on DIV21-22 and beyond. Avoid changing more than 50% of your media past DIV7 as it can kill the cells in our experience.
It could also be the attachment to the culture dish/coverslip isn't strong enough and the cells come away and die - fresh coated coverslips may help, we use Poly-D-lysine 30-70,000 Mw incubated overnight & washed thoroughly.
Good luck!
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