Neurons are very sensitive cells. So if your culture is inconsistent, first make sure that you keep conditions constant. This starts with the age of the animal (which animals are you using, which cells?), continues during preparation and ends with regular medium changes (not 100%, neurons do not like that, change 50% of the medium an add water to compensate for evaporation). This includes cell density and purity, media used and so on.

So first, try to find if any step you apply is prone to variation. Only then you can improve.

P.S.: From my experience, it is normal that from time to time primary cultures have "drop outs". So you should not react rashly, and test your conditions over several preparations. Only then try another.

Hi Simon, big thanks for your advice.

I am pretty sure that my cells conditions are constant. I culture hippocampal neuron cells from E18 Rat. About change medium, I did it, only a half of total medium. But adding the medium with water, I never try it. How you add the medium with water?

This depends on how long you really want to culture your cells. They should be fine to two weeks without adding water. How often do you change the medium?

I usually have a dish similar to the one you use for culturing with water instead of medium (no cells, of course). Depending on how many wells/dishes you have, it is easy to weigh it once per week or so to estimate the evaporation in your incubator. Keep in mind that this changes with water level of the incubator, place where you put the dishes (back/front/top/bottom), times the incubator is opened and ambient humidity and temperature and so on.

Once you know how much water is evaporating from your dishes, you can add that amount to you medium. I suggest to dilute the medium before adding it to the cells instead of putting the water directly into the dishes.

However, I do not know if many people do it like this, but I had neuronal cultures for several weeks and longer that way (human iPSC derived).

As Simon said, there is so many factors to consider - in addition to what he suggested, a few others that might help/be worth considering are: a) the coating you are using - PLL/PDL/Polyornithine/Laminin/a combination of any of the above?  In our hands (we do E16 mouse ctx neurons), PLL works best and that too the commercial BDFalcon pre-coated ones. We have tried coating our own but didn't work as well.. b) Medium type - Neurobasal Vs NBA medium; we prefer neurobasal medium  c) length of time of dissections - for us it works best to finish all dissections and start digesting within an hour of harvesting d) Method of digestion - papain Vs trypsin with DNase - papain is more gentle than trypsin in general.. Hope this helps! 

Thanks a lot for both of you, Simon and Praneeti.

Simon:

this is first time to me to consider about evaporating of culture cells by adding another water dish in incubator. I thought incubator system is enough for keep cell culture condition. That's great, so much thanks for your advice.

I change medium 3days after plating containing araC and 4 days later change medium again with fresh NB medium supplemented B27 and L-glu (only 1/3 from total medium), this refer to Banker's protocol. Then, change every 7days. Unfortunately, after 12days the cells died. 

As your information, I plating around 60.000-80.000cells/13mm coverslip. Is it enough for long term culture?

Praneeti: I've never tried PLL before. Do you think coating combination works best than single coating? I use PDL coating.

It is so hard to say one way is better than the other honestly, it works differently in different people's hands. When i was differentiating stem cells into neurons i"d use Polyornithine/laminin double-coated coverslips as polyornithine is known to promote neurite outgrowth.  I'd suggest maybe trying out a couple of different combinations or coatings with a litter of animals to get the best one. 

just use b27 +glutmax+neurobasal

1. don't use antibiotics;

2. don't use FBS;

3. don't add glutamate;

4. half change, or just add fresh medium.

my primary neuron can last more that 8 weeks, and the morphology is excellent.

Praneeti: I agree, depend on personal hands. But I think combination coating also good. I will try some variation conditions. Of course trying one by one, then I hope get the best one. :)

Hailong: one interesting reagent u mentioned is glutmax. I supplemented my neuron culture only L-glutamine (gibco). Do you completely not use FBS in your culture? I use FBS at the 2-3h after plating, then change to Neurobasal medium with supplement (B27,lglu and p/s). Btw, you don't use antibiotic? wow, excellent. No contamination until 8 weeks?.  Anyway, thanks. 

Glutmax: https://www.thermofisher.com/order/catalog/product/35050061

I don't use FBS and p/s in my culture. p/s should be the major reason for why your neuron cannot live longer.

Big thanks Hailong

Good advice above. P1-3 cultures work just as well.

Christopher: I also consider about the embryo age. Thanks :)

Also, consider the cell density. Too few cells will not survive long.

I have tried E16-e19 embryos and p0 rat cortex/hippocampus tissue. overall, the culture of embryo's brain tissue is better than p0 pups. cell density is also important, neither too high nor too low density last long. if your want to use the extraction of the cultures, cell density should be 1,000,000-2,000,000 per well (6 well plate), if the culture is for immunostaining, 20,000-50,000 per well (24 well plate) is enough.

Dmitri and Hailong: Thanks, culturing neuron should considering many factors. I vary the number of neutron, from few until high (40,000-100,000 in 24 well plate), but I was failed. Thats why, I think something wrong with my culture conditions or there are special treatments for long term culture. 

Hailong, about embryo age, some paper use P0 and p1-p3. But, I still use E18 Rat or E16.5 mice. 

Now, I am going to do some advices from all of you. Hope I am lucky!!

The standard protocol for culturing cortical neurons allows us to keep the cultures in good condition and active for months.

 Hi evryone , i have a question , after how much time i should add L-glutamine in the neurobasal/27 medium or the first  supply  with L-glutamine is sufficient and the medium can be used along the culture ( 25-28 days ) 

hi Habib..

just found your question

In my case, I add L-glutamine from the beginning in NB medium with B27.

I do not use L-glutamine, but I use glutamax.

good luck!!

hopefuly this isnt late for your experiment...

For neuronal health, co-culture with astrocytes is commonly used. However it might interfere with your experimental setup where pure populations of cells might be needed. There, CM would be beneficial. There is tons of data out there about this.

From my experience, the rule of thumb seems to be: The more heterogeneous the neuronal culture is, the more mature it is and also lives longer.

Go through the link to have best culture

https://www.sciencedirect.com/science/article/pii/S0165027016000601?via%3Dihub

Hi,

I culture E18 rat cortical neurons. I did a lot of troubleshooting to get it working and I found that the media and the plate coating made the biggest difference.

I was having adherence issues with PDL (10 ug/ml in ddH2O), the cells formed these huge clusters of bodies with floating processes connecting them. Only a few of my cultures would work and even those ones deteriorated after ~2 weeks. Adding 2% B27 (Neurobasal + 1% PenStrep + 1% Glutamine +2% B27) instead of 1% helped a lot, and doing 50% media changes once a week helped a bit too.

But the biggest difference was when switched over to dPGA from Dendrotek (https://dendrotek.ca/products/centrifuge-tube) for coating our plates. Way more consistent adherence, cultures lasted longer and seemed to have more processes on the plate. We use it the same as PDL, 10 ug/ml in ddH2O at 4C overnight. I've gotten cultures to last for 10-11 weeks using this.

Hope this helps!

Hai Ria,

Establishing a long-term culture of neuronal cells requires careful attention to several key factors. Here's a step-by-step guide to help you get started:

Remember that establishing a long-term culture of neuronal cells can be a complex process, and it may require some optimization and troubleshooting based on your specific cell line and experimental conditions. It's always a good idea to consult relevant literature, protocols, and experienced researchers in the field for guidance.

Hope it will help.

Be careful in removal of meninges during dissection and washing after trituration. This will leads to proper neurons.