Hi there,
We have some problems with (E17-18) mouse hippocampal cell cultures. After sacrificing mice (E17-18), primary neurons were cultured (400,000 cells per on the 35 mm petri dishes). For coating we use PLL and for culturing we use Neurobasal+FCS+B27 supplement+GLUTAMAX. In the cultivation we didn't observe any attachment problem with the neuronal cells in DIV1. Our cultures start to swell in DIV9-10 and die out in DIV15-16.
Does anybody have the same problem?
Thank you very much!
We use Neurobasal A (isn't it more suitable for cultures from embryos?) without FCS (->less astrocytes). Add ARAC to suppress microglia (up to 4 µM) For coating I use PDL ~50-70 µg/ml (higher concentrations require washing while could be toxic, IMHO), applied through 0.22µm filter.
Could be also a bad time. Last year we were for 2 months (Feb-March) without culture for unknown reason. Changes everything possible, nothing have helped, then everything came back to normal. Culturing neuronal cells - it's a black magic, how it was written in one of the books on this topic.
Good luck.
Hi Norbert,
for my hippocampal neuronal cultures I use PDL for coating (20 μg/ml in 1x PBS, 2h-O/N prior the preparation, 3-4 times wash with sterile water and dry).
The medium I use for plating them consists of Neurobasal medium+B27+ GlutaMAX+Pen/Strep (without FCS so that you will avoid the overgrowth of glial cells).
The density that I usually have in the 35mm dishes is a bit more than 1,000,000 cells. In my experience, the cells grow better when the culture is not too sparse, but also not too dense so that you can keep them longer. I managed to have hippocampal neurons for more than 3 weeks in culture. You could also try to add a bit of fresh medium (something like 300 μl in a total volume of 1ml) when your neurons are on DIV5-7. In this case you add a bit of nutrients without affecting the conditioned medium much so that they will have more nutrients for the next days.
Hope this helps a bit.
Good luck!
May try collagen + PDL or PLL coating (collagen overnight and 2 hrs of PDL or PLL; or as a mix of both). There are synthetic compounds that work well for coating (PEI, polyethylenimine, available from Sigma). It is important to wash the dishes well before plating. Some people add growth medium after the washing for "conditioning" the substrate. The type of the plastic could be also important; Corning 35 mm dishes work well. Alternatively, place glass coverslips into the dishes.
Do you change the culture medium during cell growth? Over the period of time in incubation, the osmolarity of the medium around the cells changes (generally increases). So if you are replacing the medium completely on day 9, it might be a shock to the cells which they dont like, swell and die. You can try to change medium half-half every 3 days while the cells are growing, its a good way to maintain osmolarity and keep the cells healthy.
Thank you very much!
I change the medium NB +B27+GLUTAMAX without FCS in DIV5. 700 ul in/200 ul out. Add CAR to suppress astroglia feeding in DIV 4-5.
And every 2 days add ca. 40-50 ul distillated water to maintain osmolarity.
Hey Norbert,
Since you don,t have any problem with cell attachment, coating seems to be fine.
If you want to have pure neuronal culture, you would want to avoid growth of glial cells.For this, I would suggest to use NB media with B27+GLUTAMAX+Pen/Strep, when you start culture.
If you are using media with FCS till DIV 5, its possible that glial cells are growing rapidly in your culture (they will not be visible) . This could be one of the reason of cell death.
Also, I would suggest to change media after DIV 7.
Best Regards
Sushma
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